Regulation of gene expression using chromatin remodelling factors

ABSTRACT

The invention provides a method to regulate expression of a gene of interest in a plant comprising, introducing into the plant a first nucleotide sequence comprising, the gene of interest operatively linked to a first regulatory region, and an operator sequence capable of binding a fusion protein, and a second nucleotide sequence comprising a second regulatory region in operative association with a nucleotide sequence encoding the fusion protein. The fusion protein comprising, a DNA binding protein, or a portion thereof, capable of binding the operator sequence, and a recruitment factor protein, or a portion thereof, capable of binding a chromatin remodelling protein. In this manner, expression of the second nucleotide sequence produces the fusion protein that regulates expression of the gene of interest.

The present invention relates to the regulation of gene expression. Moreparticularly, the present invention relates to the control of geneexpression of one or more nucleotide sequences of interest in transgenicplants using chromatin remodelling factors.

BACKGROUND OF THE INVENTION

Transgenic plants have been an integral component of advances made inagricultural biotechnology. They are necessary tools for the productionof plants exhibiting desirable traits (e.g. herbicide and insectresistance, drought and cold tolerance), or producing products ofnutritional or pharmaceutical importance. As the applications oftransgenic plants become ever more sophisticated, it is becomingincreasingly necessary to develop strategies to fine-tune the expressionof introduced genes. The ability to tightly regulate the expression oftransgenes is important to address many safety, regulatory and practicalissues. To this end, it is necessary to develop tools and strategies toregulate the expression of transgenes in a predictable manner.

Several strategies have so far been employed to control plantgene/transgene expression. These include the use of regulated promoters,such as inducible or developmental promoters, whereby the expression ofgenes of interest is driven by promoters responsive to variousregulatory factors (Gatz, 1997). Other strategies involve co-suppression(Eisner et al., 1998) or anti-sense technology (Kohno-Murase et al.,1994), whereby plants are transformed with genes, or fragments thereof,that are homologous to genes either in the sense or antisenseorientations. Chimeric RNA-DNA oligonucleotides have also been used toblock the expression of target genes in plants (Beetham et al., 1999;Zhu et al., 1999).

Posttranslational modifications of histones in chromatin are importantmechanisms in the regulation of gene expression. Protein-proteininteractions between histones H3, H4, H2A and H2B form an octomeric corewhich is wrapped with DNA. N-terminal tails of histones protrude fromthe octamer and are subject to posttranslational modification involvingacetylation and deacetylation of conserved lysine residues. A nucleosomecomprises 26 lysine residues that may be subject to acetylation.Acetylation of core histones, including H4 and H3 via histoneacetyltransferase (HAT), is correlated with transcriptionally activechromatin of eukaryotic cells. Acetylation is thought to weaken theinteractions of histones with DNA and induce alterations in nucleosomestructure. These alterations enhance the accessibility of promoters tocomponents of the transcription machinery, and increase transcription.HATs have been identified in yeast, insects, plants and mammals (e.g.Kolle et al. 1998), and are typically components of multiproteincomplexes including components of RNA polymerase II complex, TFIID,TFIIC and recruitment factors (e.g. see Lusser et al. 2001 for review).

Histone deacetylation, via histone deacetylase (HD, HDA, HDAC), isthought to lead to a less accessible chromatin conformation, resultingin the repression of transcription (e.g. Pazin and Kadonaga, 1997;Struhl, 1998; Lusser et al., 2001). The role of the yeast histonedeacetylase, RPD3, in transcriptional repression was first discoveredthrough a genetic screen for transcriptional repressors in S. cerevisiae(Vidal and Gaber, 1991). Since then, a number of yeast and mammalianHDAC genes have been cloned (Rundlett et al., 1996; Emiliani et al.,1998; Hassig. et al., 1998; Verdel and Khochbin, 1999). Most eukaryotichistone deacetylases show some sequence homology to yeast RPD3,suggesting that these proteins are all members derived from a singlegene family (Khochbin and Wolffe, 1997; Verdel and Khochbin, 1999). Inyeast and mammalian cells, the RPD3/HDACs mediate transcriptionalrepression by interacting with specific DNA-binding proteins orassociated corepressors and by recruitment to target promoters (Kadoshand Struhl, 1997; Hassig et al., 1997; Nagy et al., 1997; Gelmetti etal., 1998). Recently, a second family of histone deacetylases, HDA19 andrelated proteins, were identified in yeast and mammalian cells (Rundlettet al., 1996; Fischle et al., 1999; Verdel and Khochbin, 1999). Thedeacetylase domain of HDA19-related proteins is homologous to butsignificantly different from that of RPD3 (Fischle et al., 1999; Verdeland Khochbin, 1999). These proteins also appear to be functionallydifferent from RPD-like proteins in yeast cells (Rundlett et al., 1996).WO 97/35990 discloses mammalian-derived histone deacetylase (HDx) genesequences, gene products, and uses for these sequences and products. Thedown regulation of gene expression in plants using histone deacetylase,fused to a DNA binding domain that targeted the fusion protein to aspecific gene, has been demonstrated (Wu et al., 2000a; Wu et al.,2000b).

The present invention embraces the use of fusion proteins comprising aDNA binding domain fused to a recruitment factor, that is capable ofrecruiting chromatin remodelling proteins such as HDAC and HAT, tospecific DNA sites to regulate expression of a gene of interest. Alsodisclosed is the use of fusion proteins comprising a DNA binding portionfused to histone acetyltransferase (HAT) to regulate transcription of agene of interest.

It is an object of the invention to overcome disadvantages of the priorart.

The above object is met by the combinations of features of the mainclaims, the sub-claims disclose further advantageous embodiments of theinvention.

SUMMARY OF THE INVENTION

The present invention relates to the regulation of gene expression. Moreparticularly, the present invention relates to the control of geneexpression of one or more nucleotide sequences of interest in transgenicplants using chromatin remodelling factors.

According to an aspect of an embodiment of the present invention, thereis provided a method to regulate the expression of a gene of interest ina plant comprising:

-   -   i) introducing to the plant:        -   1) a first nucleotide sequence comprising,            -   a) the gene of interest operatively linked to a first                regulatory region,            -   b) an operator sequence capable of binding a fusion                protein, and;        -   2) a second nucleotide sequence comprising a second            regulatory region in operative association with a nucleotide            sequence encoding a fusion protein, the fusion protein            comprising,            -   a) a DNA binding protein, or a portion of a DNA binding                protein capable of binding the operator sequence, and;            -   b) a recruitment factor protein, or a portion thereof,                capable of binding a chromatin remodelling protein,    -   ii) growing the plant, wherein expression of the second        nucleotide sequence produces the fusion protein and regulates        expression of the gene of interest.

The present invention also embraces the methods as defined above,wherein the first and second regulatory regions are either the same ordifferent and are selected from the group consisting of a constitutivepromoter, an inducible promoter, a tissue specific promoter, and adevelopmental promoter.

The present invention also relates to a method of enhancing theexpression of a gene of interest or enhancing the transcription of agene of interest in a plant comprising:

-   -   i) introducing to the plant:        -   1) a first nucleotide sequence comprising,            -   a) the gene of interest operatively linked to a first                regulatory region, and;            -   b) an operator sequence that interacts with a fusion                protein;        -   2) a second nucleotide sequence comprising a second            regulatory region in operative association with a nucleotide            sequence encoding a fusion protein comprising,            -   a) a DNA binding protein, or a portion thereof, capable                of binding the operator sequence, and;            -   b) a histone acetyltransferase (HAT) protein, or portion                thereof, capable of increasing histone acetylation;    -   ii) growing the plant, wherein expression of the second        nucleotide sequence produces the fusion protein and increases        transcription of the gene of interest.

The present invention pertains to a method of regulating the expressionof a gene of interest or enhancing the transcription of a gene ofinterest in a plant comprising:

-   -   i) introducing to the plant:        -   1) a first nucleotide sequence comprising,            -   a) the gene of interest operatively linked to a first                regulatory region, and;            -   b) an operator sequence that interacts with a fusion                protein;        -   2) a second nucleotide sequence comprising a second            regulatory region in operative association with a nucleotide            sequence encoding a fusion protein comprising,            -   a) a DNA binding protein, or a portion thereof, capable                of binding the operator sequence, and;            -   b) a chromatin remodelling factor, or portion thereof,                capable of increasing histone acetylation;    -   ii) growing the plant, wherein expression of the second        nucleotide sequence produces the fusion protein and regulates        the transcription of the gene of interest.

The present invention also embraces the methods as defined above,wherein the first and second regulatory regions are either the same ordifferent and are selected from the group consisting of a constitutivepromoter, an inducible promoter, a tissue specific promoter, and adevelopmental promoter.

The first and second nucleotide sequences may be placed within the sameor within different vectors, genetic constructs, or nucleic acidmolecules. Preferably, the first nucleotide sequence and the secondnucleotide sequence are chromosomally integrated into a plant or plantcell. The two nucleotide sequences may be integrated into two differentgenetic loci of a plant or plant cell, or the two nucleotide sequencesmay be integrated into a singular genetic locus of a plant or plantcell. However, the second nucleotide sequence may be integrated into theDNA of the plant or it may be present as an extra-chromosomal element,for example, but not wishing to be limiting a plasmid.

Also, according to the present invention there is provided a method forselectively controlling the transcription of a gene of interest,comprising:

-   -   i) producing a first plant comprising a first genetic construct,        the first genetic construct comprising a first regulatory region        operatively linked to the gene of interest and at least one        operator sequence capable of binding a fusion protein;    -   ii) producing a second plant comprising a second genetic        construct, the second genetic construct comprising a second        regulatory region in operative association with a nucleic        sequence encoding the fusion protein, the fusion protein        comprising,        -   a) a DNA binding protein, or a portion thereof, capable of            binding the operator sequence, and;        -   b) a recruitment factor protein, or a portion thereof,            capable of binding a chromatin remodelling protein;    -   iii) crossing the first plant and the second plant to obtain        progeny comprising both the first genetic construct and the        second genetic construct, the progeny characterized in that the        expression of the fusion protein regulates expression of the        gene of interest.

The present invention also embraces the methods as defined above,wherein the first and second regulatory regions are either the same ordifferent and are selected from the group consisting of a constitutivepromoter, an inducible promoter, a tissue specific promoter, and adevelopmental promoter.

The present invention also pertains to the method as just defined,wherein the nucleic acid sequence encoding the fusion protein isoptimised for expression in a plant, and that the nucleotide sequenceencodes a nuclear localization signal.

Also, according to the present invention there is provided a method forselectively controlling the transcription of a gene of interest,comprising:

-   -   i) producing a first plant comprising a first genetic construct,        the first genetic construct comprising a first regulatory region        operatively linked to the gene of interest and at least one        operator sequence capable of binding a fusion protein;    -   ii) producing a second plant comprising a second genetic        construct, the second genetic construct comprising a second        regulatory region in operative association with a nucleic        sequence encoding the fusion protein comprising,        -   a) a DNA binding protein, or a portion thereof, capable of            binding the operator sequence, and;        -   b) a HAT protein, or portion thereof, capable of histone            acetylation in plants;    -   iii) crossing the first plant and the second plant to obtain        progeny comprising both the first genetic construct and the        second genetic construct and characterized in that the        expression of the fusion protein up-regulates the expression of        the gene of interest.

The present invention also provides the method as just defined, wherein,the nucleic acid sequence encoding the fusion protein is optimised forexpression in the plant, and that the nucleic acid sequence encodes anuclear localization signal.

The present invention also embraces the methods as defined above,wherein the first and second regulatory regions are either the same ordifferent and are selected from the group consisting of a constitutivepromoter, an inducible promoter, a tissue specific promoter, and adevelopmental promoter.

Furthermore, this invention provides a method to regulate expression ofan endogenous nucleic acid sequence of interest in a plant comprising:

-   -   i) introducing into the plant a nucleotide sequence comprising,        a regulatory region, operatively linked with a nucleotide        sequence encoding a fusion protein, the fusion protein        comprising,        -   a) a DNA binding protein, or a portion thereof, capable of            binding a segment of a DNA sequence of the endogenous            nucleotide sequence of interest;        -   b) a recruitment factor protein, or a portion thereof,            capable of binding a chromatin remodelling protein; and    -   ii) growing the plant, wherein expression of the nucleotide        sequence produces the fusion protein that regulates expression        of the endogenous nucleic acid sequence of interest.

The present invention also includes a method to regulate expression ofan endogenous nucleic acid sequence of interest in a plant comprising:

-   -   i) introducing into the plant a nucleotide sequence comprising a        regulatory region, operatively linked with a nucleotide sequence        encoding a recruitment factor protein, the recruitment factor        protein capable of binding an endogenous DNA binding protein,        the endogenous DNA binding protein characterized in binding a        segment of a DNA sequence of the endogenous nucleotide sequence        of interest, and;    -   ii) growing the plant, wherein expression of the nucleotide        sequence produces the recruitment factor thereby regulating        expression of the endogenous nucleic acid sequence of interest.

This summary of the invention does not necessarily describe allnecessary features of the invention but that the invention may alsoreside in a sub-combination of the described features.

BRIEF DESCRIPTION OF THE DRAWINGS

These and other features of the invention will become more apparent fromthe following description in which reference is made to the appendeddrawings wherein:

FIG. 1 shows the nucleotide and deduced amino acid sequences of wildtype ROS and a modified ROS of Agrobacterium tumefaciens. FIG. 1(A)shows the amino acid sequence alignment of known ROS repressors(wild-type ROS, SEQ ID NO:1; ROSR, SEQ ID NO:63; ROSAR, SEQ ID NO: 64;MucR, SEQ ID NO: 65), and a synthetic ROS (SEQ ID NO: 4). The amino acidsequence ‘PKKKRKV’ (SEQ ID NO: 6) at the carboxy end of synthetic ROS isone of several nuclear localization signals. FIG. 1(B) shows thenucleotide sequence of a synthetic ROS (SEQ ID NO:2) that had beenoptimised for plant codon usage containing a nuclear localization signalpeptide (in italics). Optional restriction sites at the 5′ end of thesequence are underlined. FIG. 1(C) shows the consensus nucleotide (SEQID NO:3) and predicted amino acid (SEQ ID NO:4) sequence, of a compositeROS sequence comprising all possible nucleotide sequences that encodewild type ROS repressor, and the wild type ROS amino acid sequence. Theamino acid sequence ‘PKKKRKV’ (SEQ ID NO:6) at the carboxy endrepresents a nuclear localization signal. Amino acids in bold identifythe zinc finger motif. Nucleotide codes are as follows: N=A or C or T orG; R=A or G; Y=C or T; M=A or C; K=T or G; S=C or G; W=A or T; H=A or Tor C; B=T or C or G; D=A or T or G; V=A or C or G. FIG. 1(D) shows thenucleotide sequence of the operator sequences of the virC/virD (SEQ IDNO:27) and ipt (SEQ ID NO:8) genes. FIG. 1(E) shows a consensus operatorsequence (SEQ ID NO:5) derived from the virC/virD (SEQ ID NOs:66-67) andipt (SEQ ID NOs:68-69) operator sequences. This sequence comprises 10amino acids, however, only the first 9 amino acids are required forbinding ROS.

FIG. 2-4 shows in a diagrammatic form several variations of regulatinggene expression using the methods of the present invention.

FIG. 5 shows schematic representations of nucleotide constructs thatplace the expression of a gene of interest under the control aregulatory region, in this case a CaMV35S regulatory region, modified tocontain a ROS operator site. FIG. 5(A) shows the nucleotide constructp74-315 in which a CaMV35S regulatory region, modified to contain a ROSoperator site downstream of the TATA box, is operatively linked to agene of interest (β-glucuronidase; GUS). FIG. 5(B) shows the nucleotideconstruct p74-316 in which a CaMV35S regulatory region is modified tocontain a ROS operator site upstream of the:TATA box is operativelylinked to the protein encoding region of GUS. FIG. 5(C) shows thenucleotide construct p74-309 in which a CaMV35S regulatory regionmodified to contain ROS operator sites upstream and downstream of theTATA box is transcriptionally fused (i.e. operatively linked) to theprotein encoding region of GUS. FIG. 5(D) shows construct p74-118comprising a 35S regulatory region with three ROS operator sitesdownstream from the TATA box. The 35S regulatory region is operativelylinked to the gene of interest (GUS).

FIG. 6 shows a schematic representation of a nucleotide construct thatplaces the expression of a gene of interest gene under the control of aregulatory region, in this case, the tms2 regulatory region that hasbeen modified to contain ROS operator sites. FIG. 6(A) shows thenucleotide construct p76-507 in which a tms2 regulatory region isoperatively linked to a gene of interest (in this case encodingβ-glucuronidase, GUS). FIG. 6(B) shows the nucleotide construct p76-508in which a tms2 regulatory region modified to contain two tandemlyrepeated ROS operator sites downstream of the TATA box istranscriptionally fused (i.e. operatively linked) to the protein codingregion of GUS.

FIG. 7 shows a schematic representation of a nucleotide construct thatplaces the expression of a gene of interest under the control of aregulatory region, in this case actin 2 regulatory region, that has beenmodified to contain ROS operator sites. FIG. 7(A) shows the nucleotideconstruct p75-101 in which an actin2 regulatory region is operativelylinked to a gene of interest (the β-glucuronidase (GUS) reporter gene).FIG. 7(B) shows the nucleotide construct p74-501 in which an actin2regulatory region modified to contain two tandemly repeated ROS operatorsites upstream of the TATA box is transcriptionally fused (operativelylinked) to the a gene of interest (GUS).

FIG. 8 shows Southern analysis of transgenic Arabidopsis plants. FIG.8(A) shows Southern analysis of a plant comprising a first geneticconstruct, p74-309 (35S-operator sequence-GUS; see FIG. 5(C) for map).DNA was digested with ClaI or XhoI and the blot was probed with the ORFof the GUS gene. FIG. 8(B) shows Southern analysis of a plant comprisinga second genetic construct p75-101 (see FIG. 7A). HindIII digests wereprobed with NPTII.

FIG. 9 shows expression of a gene of interest in plants. Upper panelshows expression of GUS under the control of 35S (pBI121; 35S:GUS).Middle panel shows GUS expression under the control of actin2 comprisingROS operator sequences (p74-501; see FIG. 7(B) for construct). Lowerpanel shows the lack of GUS activity in a non-transformed control.

FIG. 10 shows alignments of bnKCP1 and sequence comparison of kinaseinducible domains (KIDs) in bnKCP1 and CREB family members. FIG. 10(A)shows alignment of the deduced amino acid sequences of bnKCP1 (SEQ IDNO:71), atKCP (SEQ ID NO:72), atKCL1 (SEQ ID NO:73) and atKCL2 (SEQ IDNO:74) proteins. Serine (S)-rich residues and the conserved region(GKSKS domain) among the four sequences are single underlined and doubleunderlined, respectively. The putative nuclear localization signal (NLS)and the phosphorylation site of protein kinase A are indicated byasterisks and diamonds, respectively. FIG. 10(B) shows alignment of theamino acid sequences of bnKCP1 (SEQ ID NO:75), hydra CREB (hyCREB). (SEQID NO:77), canfa CREM (cCREM) (SEQ ID NO:80), and mammalian ATF-1 (SEQID NO:76), CREB (SEQ ID NO:78) and CREM (SEQ ID NO:79). Diamondsindicate the conserved phosphorylation site of protein kinase A. FIG.10(C) shows a phylogenetic tree of the KIDs sequences using the NTIVector program.

FIG. 11 shows structural features of bnKCP1. FIG. 11(A) shows schematicrepresentation of entire bnKCP1 protein. Numbers above or under theboxes refer to positions of amino acid residues. S-rich (34-58), GKSKS(88-143) and KID (161-215) domains or motifs are shown in dotted boxes,the nuclear localization signal (NLS) in black box, and the three acidicmotifs (I, II, III) in gray boxes. FIG. 11(B) shows secondary structurefeatures and hydrophilicity of bnKCP1 analyzed using DNAstar Proteanprogram.

FIG. 12 shows Southern blot analysis of Brassica genomic DNA. Totalgenomic DNA (10 μg/lane) from Brassica napus cv Westar was digested withrestriction enzymes EcoRI (EI), XbaI (X), HindIII (H), PstI (P), EcoRV(EV) and KpnI (K). The entire ORF of bnKCP1 was used as a probe.

FIG. 13 shows in vitro interaction of wild type and mutant bnKCP1proteins with the GST-HDA19 and GST-Gcn5 fusion proteins. FIG. 13(A)shows a schematic representation of the bnKCP1 and its deletion mutantsobtained by deletion of C-terminal regions of bnKCP1. FIG. 13(B) showsbinding activities of bnKCP1 and its mutants with GST-HDA19, GST-Gcn5and GST alone (negative control), respectively, as indicated. The wildtype bnKCP1, mutants bnKCP1¹⁻¹⁶⁰ and bnKCP1¹⁻⁸⁰, luciferase (as positivecontrol) and negative control (no template) were produced using in vitrotranscription/translation reactions. The translation products wereincubated with GST fusion proteins or GST and their binding activitieswere examined as described in Example 4. FIG. 13(C) shows activation oflacZ reporter gene by bnKCP1 and its deletion mutants, ΔbnKCP1¹⁻¹⁶⁰ andΔbnKCP1¹⁻⁸⁰, in yeast cells. MaV203 yeast cells carrying plasmidpDBLeu-HDA19 and the reporter gene were transfected with the plamidpPC86-bnKCP1, pPC86-bnKCP1¹⁻¹⁶⁰, pPC86-bnKCP1¹⁻⁸⁰ or pPC86 vector only.Yeast strains A and B were used as negative and positive controls,respectively. The β-galactosidase activity was assayed usingchlorophenol red-β-D-galactopyranoside (CPRG) and was expressed as apercentage of activity conveyed by bnKCP1.

FIG. 14 shows the effect of S¹⁸⁸ on the interaction between bnKCP1 andGST-HDA19 fusion protein. A glycine residue (G¹⁸⁸) was introduced bysite-directed mutagenesis to replace S¹⁸⁸. The binding activities ofwild-type bnKCP1 and the mutant ΔbnKCP1¹⁸⁸ with GST-HDA19 or GST alone(negative control) were examined with GST pulldown affinity assay asdescribed in Example 4. FIG. 14A shows the introduction of G188 into theKID of bnKCP1. FIG. 14B shows in vitro protein interaction of bnKCP1 andthe mutant ΔbnKCP1G¹⁸⁸ with GST-HDA19 or GST alone.

FIG. 15 shows expression patterns of bnKCP1 mRNA in different tissues.Total RNA (20 μg/lane) was isolated from leaves with petioles, flowers,roots, stems and immature siliques.

FIG. 16 shows expression of bnKCP1 gene in response to low temperature,LaCl₃ and inomycin treatments. Total RNA (20 μg/lane) was isolated fromleaf blades of four-leaf stage Brassica napus cv Westar seedlings afterexposure to different stress conditions and analyzed by northernblotting using the bnKCP1 ORF as probe. FIG. 16(A) shows bnKCP1transcript accumulation in leaves and stems of seedlings exposed to cold(4° C.). FIG. 16(B) shows expression pattern of bnKCP1 gene aftertreatment with LaCl₃ and inomycin.

FIG. 17 shows transactivation, of the lacZ gene by bnKCP1 in yeast. ThelacZ gene was driven by a promoter containing GAL4 DNA binding sites andintegrated into the genome of yeast MaV203. FIG. 17(A) is a schematicrepresentation of the bnKCP1 and its deletion mutants. FIG. 17(B) Yeastcells carrying the reporter gene were transfected with the effectorplasmids pDBLeu-bnKCP1, pDBLeu-bnKCP1¹⁻¹⁶⁰, pDBLeu-bnKCP1¹⁻⁸⁰, andpDBLeu-bnKCP1⁸¹⁻²¹⁵ or the pDBLeu vector only. Yeast strains A and B(GibcoL BRL, Life Technologies) were used as negative and positivecontrols, respectively. The β-galactosidase activity was assayed usingCPRG (chlorophenol red-β-D-galactopyranoside) and was expressed as apercentage of activity conveyed by the positive control (strain C). Barsindicate the standard error of three replicates.

FIG. 18 shows the nuclear localization of GUS-bnKCP1 protein in onioncells. FIG. 18(A) is a schematic diagram of the GUS-bnKCP1 fusionconstruct containing the CaMV 35S promoter. The bnKCP1 was fusedin-frame to the GUS reporter gene. FIG. 18(B) shows transient expressionof GUS-bnKCP1 fusion protein (top) and GUS alone (bottom) in onioncells. Onion tissues were simultaneously analysed using histochemicalGUS assay (left) and nucleus-specific staining with DAPI (right) asdescribed in Example 4.

FIG. 19 shows a diagrammatic representation of a strategy for preparingfusions between a recruitment factor involved in chromatin remodellingand a DNA binding protein. In the non-limiting example shown in thisfigure, the recruitment factor is KID (see Example 4), and the DNAbinding protein is a zinc finger.

FIG. 20 shows alignment of the deduced products of BnSCL1 (SEQ IDNO:81), AtSCL15 (accession number Z99708) (SEQ ID NO:82) and LsSCL(accession number AF273333) (SEQ. ID NO:83). Identical and conservedamino acids in the three proteins are shown as white letters on a blackbackground and black letter on a gray background, respectively. Aminoacids with weak similarity are indicated as white letters on a graybackground. Amino acids with no similarity are shown as black letters ona white background. The putative nuclear localization signals and LXXLLmotif are indicated by asterisks and dots, respectively. The VHIIDmotif, two leucine heptad regions (LHRI and LHRII), PFYRE and SAW motifare underlined as indicated.

FIG. 21 shows a phylogenetic tree of the GRAS family sequences made bythe NTI Vector program in Brassica napus, Arabidopsis thaliana, Hordeumvulgare, Zea mays, Lycopersicon esculentum, Pisum sativum and Oryzasativa. The BnSCL1 is underlined.

FIG. 22 shows DNA gel blot analysis of BnSCL1 gene. Total genomic DNA(10 μg/lane) from Brassica napis was digested with restriction enzymesEcoRI (ED), XbaI (X), HindIII (H), PstI (P), EcoRV (EV) and KpnI (K),and hybridized with the entire ORF of BnSCL1 under high stringencyconditions.

FIG. 23 shows in vitro interaction of wild type and mutant BnSCL1proteins with the GST-HDA19 fusion protein. FIG. 23(A) is a schematicrepresentation of the BnSCL1 and its deletion mutants obtained by thedeletion of its C-terminal regions. FIG. 23(B) shows the bindingactivities of BnSCL1 and its mutants with GST-HDA19. The wild typeBnSCL1, mutants ΔBnSCL1¹⁻³⁵⁸, ΔBnSCL1¹⁻²⁶¹, ΔBnSCL1¹⁻²¹⁷ andΔBnSCL1¹⁻¹⁴⁵, luciferase (positive control) and negative control (notemplate) were produced using in vitro transcription/translationreactions. The translation products were incubated with GST fusionproteins or GST alone (data not shown) and their binding activities wereexamined as described in Example 5. Arrow point to band representing thein vitro translated ΔBnSCL1¹⁻¹⁴⁵ protein that did not bind to therecombinant protein.

FIG. 24 shows in vivo interaction of wild type and mutant BnSCL1proteins. FIG. 24(A) is a schematic representation of the BnSCL1 and itsdeletion mutants. FIG. 24(B) shows the activation of lacZ reporter geneby BnSCL1 and its deletion mutants in yeast cells. MaV203 yeast cellscarrying plasmid pDBLeu-HDA19 and the lacZ reporter gene weretransfected with the plasmid pPC86-BnSCL1, pPC86-BnSCL1¹⁻³⁵⁸,pPC86-BnSCL1¹⁻²⁶¹, pPC86-BnSCL1¹⁻²¹⁷, pPC86-BnSCL1¹⁻¹⁴⁵,pPC86-BnSCL1¹⁴⁶⁻³⁵⁸, pPC86-BnSCL1²¹⁸⁻⁴³⁸ or pPC86 vector only. Thenegative control yeast strain A, and the positive controls yeast strainsB and C (GIBCOL BRL, Life Technologies) were also used. Theβ-Galactosidase activity was assayed using CPRG (chlorophenolred-β-D-galactopyranoside) and was expressed as a percentage of activityconveyed by yeast strain C. Bars indicate the standard error of threereplicates.

FIG. 25 shows transactivation of the lacZ gene by BnSCL1 protein inyeast. FIG. 25(A) is a schematic representation of the BnSCL1 and itsdeletion mutants. FIG. 25(B) shows the activation of lacZ reporter geneby BnSCL1 and its deletion mutants in yeast cells. The lacZ reportergene was driven by a promoter containing GAL4 DNA binding sites andintegrated into the genome of yeast MaV203 cell. Yeast cells carryingthe reporter gene were transfected with the effector plasmidspDBLeu-BnSCL1, pDBLeu-BnSCL1¹⁻³⁵⁸, pDBLeu-BnSCL1¹⁻²⁶¹,pDBLeu-BnSCL1¹⁻²¹⁷, pDBLeu-BnSCL1¹⁻¹⁴⁵, pDBLeu-BnSCL1¹⁴⁶⁻³⁵⁸,pDBLeu-BnSCL1²¹⁸⁻⁴³⁸ or pDBLeu vector only. Yeast strains A, B, C and D(GIBCOL BRL, Life Technologies) were used as controls as described inExample 5. The β-Galactosidase activity was assayed using CPRG and wasexpressed as a percentage of activity conveyed by the wild type BnSCL1protein. Bars indicate the standard error of three replicates.

FIG. 26 shows expression patterns of BnSCL1 mRNA in different tissues.FIG. 26(A) is a RNA gel blot analysis of total RNA (20 μg/lane) isolatedfrom leaves, flowers, roots, stems and siliques, electrophoresed througha 1.2% agarose gel containing formaldehyde and probed with the ORF ofBnSCL1 as described in Example 5. EtBr stained total RNA is shown toindicate even loading. FIG. 26(B) is a quantitative one-step RT-PCRanalysis of total RNA extracted from leaves, flowers, roots, stems,siliques and shoots. Quantitative RT-PCR products were electrophoresedthrough a 1% agarose gel and hybridized with ³²P-labelled 5′-endfragment (435 bp) of BnSCL1 ORF. A 960 bp fragment of the Brassica napusactin gene co-amplified with BnSCL1 was used as an internal standard asdescribed in Example 5.

FIG. 27 shows expression of BnSCL1 gene in four-leaf stage Brassicanapus seedlings in the presence or absence of 2,4-D. Total RNA wasisolated from the fourth leaves after the indicated period of the firstfoliar application of 1 mM 2,4-D and subjected to quantitative one-stepRT-PCR. The RT-PCR products were analyzed by Southern blotting using theBnSCL1 ORF as probe (left) and the blotting results were showngraphically relative to the level of internal standard Actin (arbitraryvalue of 100)(right).

FIG. 28 shows kinetics of BnSCL1 mRNA accumulation in response to auxinin the presence and absence of histone deacetylase inhibitor sodiumbutyrate. Nine-day-old light-grown seedlings were treated with 10 mMsodium butyrate for 24 h followed by exogenous 2,4-D application atvariable concentrations as indicated. Quantitative one-step RT-PCR wasused to analyze total RNA extracted from shoots (FIG. 28A) and roots(FIG. 28B) (see legend to FIG. 27 Expression of BnSCL1 in response to2,4-D was also analyzed using quantitative RT-PCR of total RNA isolatedfrom shoots and roots of 10 dpg seedlings in the presence of 50 μM NPA,an auxin transport inhibitor, for 24 h before the exogenous applicationof 2,4-D (FIG. 28C).

FIG. 29 shows in a diagrammatic form several constructs that may be usedto regulate gene expression as described in Example 6.

DESCRIPTION OF PREFERRED EMBODIMENT

The present invention relates to the regulation of gene expression. Moreparticularly, the present invention relates to the control of geneexpression of one or more nucleotide sequences of interest in transgenicplants using chromatin remodelling factors.

The following description is of a preferred embodiment by way of exampleonly and without limitation to the combination of features necessary forcarrying the invention into effect.

Gene regulation can be used in applications such as metabolicengineering to produce plants that accumulate large amounts of certainintermediate compounds. Regulation of gene expression can also be usedfor control of transgenes across generations, or production of F1 hybridplants with seed characteristics that would be undesirable in theparental line, for example but not limited to, hyper-high oil, reducedfiber content, low glucosinolate levels, reduced levels of phytotoxins,and the like. In the latter examples, low glucosinolate levels, or otherphytotoxins, may be desired in seeds while higher concentrations ofthese compounds may be required elsewhere, for example in the case ofglucosinolates, within cotyledons, due to their role in plant defence.Another non-limiting example for the controlled regulation of a gene ofinterest during plant development is seed specific down regulation ofsinapine biosynthesis, as for example in seeds of Brassica napus. Inmany instances, transgene expression needs to be regulated only incertain plant organs/tissues or at certain stages of development. Themethods as described herein may also be used to control the expressionof a gene of interest that encodes a protein used to for plant selectionpurposes. For example, which is to be considered non-limiting, a gene ofinterest may encode a protein that is capable of metabolizing a compoundfrom a non-toxic form to a toxic form thereby selectively removingplants that express the gene of interest.

The present invention provides a method to regulate the expression of agene of interest by transforming a plant with one or more constructscomprising:

-   -   1) a first nucleotide sequence comprising,        -   a) a nucleic acid sequence of interest operatively linked to            a regulatory region,        -   b) an operator sequence capable of binding a fusion protein,            and;    -   2) a second nucleotide sequence comprising a regulatory region        in operative association with a nucleotide sequence encoding a        fusion protein, the fusion protein comprising,        -   a) a DNA binding protein, or a portion of a DNA binding            protein capable of binding the operator sequence, and;        -   b) a recruitment factor protein, or a portion of a            recruitment factor protein capable of binding a chromatin            remodelling protein,            wherein binding of the fusion protein to the operator            sequence of the first nucleotide sequence regulates            expression of the nucleic acid sequence of interest from the            first nucleotide sequence. The operator sequence of the            first nucleotide sequence may be positioned upstream of the            ORF of the nucleic acid sequence of interest.

These first and second nucleotide sequences may be placed within thesame or within different vectors, genetic constructs, or nucleic acidmolecules. Preferably, the first nucleotide sequence and the secondnucleotide sequence are chromosomally integrated into a plant or plantcell. The two nucleotide sequences may be integrated into two differentgenetic loci of a plant or plant cell, or the two nucleotide sequencesmay be integrated into a singular genetic locus of a plant or plantcell. However, the second nucleotide sequence may be integrated into theDNA of the plant or it may be present as an extra-chromosomal element,for example, but not wishing to be limiting a plasmid.

By “operatively linked” it is meant that the particular sequencesinteract either directly or indirectly to carry out their intendedfunction, such as mediation or modulation of gene expression. Theinteraction of operatively linked sequences may, for example, bemediated by proteins that in turn interact with the sequences. Atranscriptional regulatory region and a sequence of interest are“operably linked” when the sequences are functionally connected so as topermit transcription of the sequence of interest to be mediated ormodulated by the transcriptional regulatory region.

By the term “regulate the expression” it is meant reducing or increasingthe level of mRNA, protein, or both mRNA and protein, encoded by a geneor nucleotide sequence of interest in the presence of the fusion proteinencoded by the second nucleotide sequence, relative to the level ofmRNA, protein or both mRNA and protein encoded by the nucleic acidsequence of interest in the absence of the fusion protein encoded by thesecond nucleotide sequence.

By the term “fusion protein” it is meant a protein comprising two ormore amino acid portions which are not normally found together withinthe same protein in nature and that are encoded by a single gene. Fusionproteins may be prepared by standard techniques in molecular biologyknown to those skilled in the art (see for example FIG. 17). In thecontext of the present invention, at least one of the amino acidportions is capable of binding an operator sequence as defined herein.

By the term “binding” it is meant reversible or non-reversibleassociation of two components, for example the operator sequence and theDNA binding domain of a protein, including a fusion protein, or therecruitment factor protein and chromatin remodelling protein asdescribed herein. Preferably, the two components have a tendency toremain associated, but are capable of dissociation under appropriateconditions. Conditions may include, but are not limited to the additionof a third component, chemical, etc which enhances dissociation of thebound components.

By the term “recruitment factor” it is meant a protein or peptidesequence capable of interacting with, or binding a chromatin remodellingprotein. Preferably, the recruitment factor and the chromatinremodelling protein interact or bind in a manner such that the activityof the chromatin remodelling protein is retained. However, by bindingthe recruitment factor, the activity of the chromatin remodellingprotein may be modified in some manner. Non-limiting examples ofrecruitment factors include KID, for example bnKCP1, or fragmentsthereof (Example 4), BnSCL1, or fragments therof (Example 5), ADA, SAGA,STAGA, PCAF, TFIID, and TFIIIC (Lusser, 2001, Table 1, which isincorporated herein by reference). A recruitment factor may be modifiedto include a DNA binding region, for example as outlined in FIG. 17, [ora native recruitment factor may be utilized to target proteins thatinteract with genes in their native context]. An example, which is notto be considered limiting in any manner, bnKCP1, or active fragmentsthereof (see Example 4) can target transcription factors that are knownto bind DNA. Examples of such transcription factors include ERF (Hart etal., 1993), SEBF (Boyle and Brisson, 2001), or CBF (Stockinger et al.,1997). In this manner by over expressing bnKCP1, regulation of theexpression of a gene that is dependant on ERF, CBP or SEBF activity maybe regulated. Another non-limiting example of a recruitment factor isBnSCL1, or active fragments thereof (see Example 5). An example, whichis not to be considered limiting, of a protein that interacts withbnKCP1 and BnSCL1 is the chromatin remodelling protein HDAC, for exampleHDA19.

By the term “chromatin remodelling protein” it is meant a protein thatis capable of altering the structure of chromatin. Preferably thechromatin remodelling protein is histone acetyl transferase (HAT) orhistone deacetylase (referred to either as HD, HDA, or HDAC). Any HATprotein, HDAC protein, or any derivative of any HAT protein or HDACprotein may be used in the method of the present invention provided thatthe HAT protein, HDAC protein or derivative thereof exhibits therespective histone acetylase, or histone deacetylase activity in plants.

By the term “HD binding domain” or “histone deacetylase binding domain”,it is meant a sequence of amino acid residues which interacts with ahistone deacetylase enzyme through protein-protein interactions. Suchprotein-protein interactions can be monitored in several ways, forexample, which is not to be considered limiting, by yeast two-hybridexperiments. Non-limiting examples of proteins comprising a HD bindingdomain include bnKCP1 and BnSCL1.

By the term “DNA binding protein or portion of a DNA binding protein” itis meant a protein or amino acid sequence capable of binding to aspecific operator sequence. By “operator sequence” it is meant asequence of DNA that is capable of binding to the DNA binding protein orportion of the DNA binding protein. Examples of a DNA binding proteinscapable of binding specific operator sequences include, but are notlimited to, the ROS repressor, TET repressor, Sin3, VP16, GAL4, Lex A,UMe6, ERF, SEBF and CBF. Any DNA binding protein or portion of any DNAbinding protein may be employed in the method of the present inventionprovided that the protein or portion thereof is capable of binding to anoperator sequence. As an example, but not to be considered limiting inany manner, the ROS repressor may be employed in the method of thepresent invention. By ROS repressor it is meant any ROS repressor,analog or derivative thereof as known within the art which is capable ofbinding to an operator sequence. These include ROS repressors asdescribed herein, as well as other microbial ROS repressors, for examplebut not limited to ROSAR (Agrobacterium radiobacter; Brightwell et al.,1995) (SEQ ID NO:64), MucR (Rhizobium meliloti; Keller M et al., 1995)(SEQ ID NO:65), and ROSR (Rhizobium elti; Bittinger et al., 1997; alsosee Cooley et al., 1991; Chou et al., 1998; Archdeacon J et al., 2000;D'Souza-Ault M. R., 1993; all of which are incorporated herein byreference) (SEQ ID NO:63). The DNA sequence of ROS, or any other DNAbinding protein, may be modified to optimize expression within a plant.Examples of ROS repressors that may be used as described herein areprovided in FIGS. 1(A) to (C) and (SEQ ID NOs: 1-4).

The DNA binding protein, or portion thereof that exhibits DNA bindingactivity may be fused to a recruitment factor or chromatin remodellingprotein as described herein. Examples of such fusion proteins can beprepared, using methods known in the art, for example but not limited tothe method outlined in FIG. 17. FIG. 17 discloses a strategy forcreating fusion between the zinc finger domain of the ROS repressor andthe KID domain of bnKCP1. This involves amplification of regionsencoding the zinc finger domain of the ROS repressor and the KID domainusing the following primers:

-   zinc finger: The forward primer (zf-F) contains a restriction enzyme    site at the 5′ end and the reverse primer (zf-R) contains 15    nucleotides from the 5′ end of the KID region.;-   KID domain: The forward primer (KID-F) contains 15 nucleotides from    the 3′ region of the zinc finger domain, and the reverse primer    (KID-R) contains a restriction enzyme site at the 3′ end.    The amplified zinc finger and KID fragments are combined and used as    a template for a new round of PCR amplification where only the    forward primer (zf-F) of the zinc finger and the reverse primer    (KID-R) of the KID domain are used. The two separate templates are    amplified to create one single in frame fusion fragment encoding the    zinc finger and KID domains, and containing restriction sites at    each end. This product is then cloned into a plant expression    vector.

However, it is to be understood that fusion of a recruitment factor witha DNA binding protein may not be required in order to regulateexpression of a nucleic acid sequence of interest. Recruitment factorsare known to bind chromatin remodelling proteins and factors thatdirectly or indirectly bind DNA. For example, bnKCP1 (Example 4)exhibits the property of binding ERF.

Depending upon the chromatin remodelling protein selected, geneexpression may be up-regulated or down-regulated. For example, which isnot to be considered limiting in any manner, the binding of a fusionprotein containing a recruitment factor capable of recruiting HAT to agene, may result in up-regulation of expression of a nucleic acidsequence of interest, while a fusion protein that recruits HDAC willresult in the down-regulation of the expression of a nucleic acidsequence of interest. However, it is within the scope of the presentinvention that modification to the rate of up-regulation anddown-regulation of gene expression may occur depending upon the locationof the operator sequence that binds the fusion protein.

The operator sequence is preferably located in proximity to the nucleicacid sequence of interest, either upstream of or downstream of thenucleic acid sequence of interest (see for example FIG. 5A-D).Alternatively, the operator sequence may be within the non-coding regionof the nucleic acid sequence of interest, for example, but not wishingto be limiting, within an intron of the gene. If it is desired to havethe expression of a nucleic acid sequence of interest reduced orrepressed, the operator sequence may be located within a nucleotideregion that interferes with binding of transcription factors requiredfor transcription of the nucleic acid sequence of interest, for example,interfering with the binding of the RNA polymerase to the nucleic acidsequence of interest, or reducing the rate of migration of thepolymerase along a nucleotide sequence, or both.

An operator sequence may consist of a minimal sequence required forbinding of a DNA binding protein or fragment thereof, or it may comprisean inverted repeat or palindromic sequences of a specified length. Forexample, but not wishing to be limiting, the ROS operator sequence maycomprise 9 or more nucleotide base pairs (see FIGS. 1(D) and (E)) thatexhibits the property of binding a DNA binding domain of a ROSrepressor. A consensus sequence of a 10 amino acid region including the9 amino acid DNA binding site sequence is WATDHWKMAR (SEQ ID NO: 5; FIG.1(E)). The last amino acid, “R”, of the consensus sequence is notrequired for ROS binding (data not presented). Examples of operatorsequences, which are not to be considered limiting in any manner, alsoinclude, as is the case with the ROS operator sequence from the virC orvirD gene promoters, a ROS operator made up of two 11 bp invertedrepeats separated by TTTA:

TATATTTCAATTTTATTGTAATATA; (SEQ ID NO: 7) orthe operator sequence of the IPT gene:

TATAATTAAAATATTAACTGTCGCATT. (SEQ ID NO: 8)However, it is to be understood that analogs or variants of SEQ IDNO's:7, 8 and 5 may also be used providing they exhibit the property ofbinding a DNA binding domain, preferably a DNA binding domain of the ROSrepressor. For example, but not to be considered limiting in any manner,in the promoter of the divergent virC/virD genes of Agrobacteriumtumefaciens, ROS binds to a 9 bp inverted repeat sequence in anorientation-independent manner (Chou et al., 1998). The ROS operatorsequence in the ipt promoter also consists of a similar sequence to thatin the virC/virD except that it does not form an inverted repeat (Chouet al., 1998). Only the first 9 bp are homologous to ROS box invirC/virD indicating that the second 9 bp sequence may not be arequisite for ROS binding. Accordingly, the use of ROS operatorsequences or variants thereof that retain the ability to interact withROS, as operator sequences to selectively control the expression ofgenes or nucleotide sequences of interest, is within the scope of thepresent invention.

Other operator sequences include sequences known to bind transcriptionfactors, for example but not limited to:

-   -   TAAGAGCCGCC (SEQ ID NO:9), which is known to bind ERF (in        ethylene responsive genes; Hart et al., 1993);    -   GACTGTCAC (SEQ ID NO:10), which is known to bind to SEBF (in        pathogenesis responsive genes; Boyle and Brisson, 2001);        -   TACCGACAT (SEQ ID NO:11) and TGGCCGAC (SEQ ID NO:12), which            are known to bind CBF (in low temperature responsive genes;            Stockinger et al., 1997).            The transcription factors ERF, SEBF and CBF are example of            factors that can be targeted by the recruitment factor            bnKCP1.

By “regulatory region” or “regulatory element” it is meant a portion ofnucleic acid typically, but not always, upstream of the protein codingregion of a gene, which may be comprised of either DNA or RNA, or bothDNA and RNA. When a regulatory region is active and in operativeassociation with a nucleic acid sequence of interest, this may result inexpression of the nucleic acid sequence of interest. A regulatoryelement may be capable of mediating organ specificity, or controllingdevelopmental or temporal gene activation. A “regulatory region”includes promoter elements, core promoter elements exhibiting a basalpromoter activity, elements that are inducible in response to anexternal stimulus, elements that mediate promoter activity such asnegative regulatory elements or transcriptional enhancers. “Regulatoryregion”, as used herein, also includes elements that are activefollowing transcription, for example, regulatory elements that modulategene expression such as translational and transcriptional enhancers,translational and transcriptional repressors, upstream activatingsequences, and mRNA instability determinants. Several of these latterelements may be located proximal to the coding region.

In the context of this disclosure, the term “regulatory element” or“regulatory region” typically refers to a sequence of DNA, usually, butnot always, upstream (5′) to the coding sequence of a structural gene,which controls the expression of the coding region by providing therecognition for RNA polymerase and/or other factors required fortranscription to start at a particular site. However, it is to beunderstood that other nucleotide sequences, located within introns, or3′ of the sequence may also contribute to the regulation of expressionof a coding region of interest. An example of a regulatory element thatprovides for the recognition for RNA polymerase or other transcriptionalfactors to ensure initiation at a particular site is a promoter element.Most, but not all, eukaryotic promoter elements contain a TATA box, aconserved nucleic acid sequence comprised of adenosine and thymidinenucleotide base pairs usually situated approximately 25 base pairsupstream of a transcriptional start site. A promoter element comprises abasal promoter element, responsible for the initiation of transcription,as well as other regulatory elements (as listed above) that modify geneexpression.

There are several types of regulatory regions, including those that aredevelopmentally regulated, inducible or constitutive. A regulatoryregion that is developmentally regulated, or controls the differentialexpression of a gene under its control, is activated within certainorgans or tissues of an organ at specific times during the developmentof that organ or tissue. However, some regulatory regions that aredevelopmentally regulated may preferentially be active within certainorgans or tissues at specific developmental stages, they may also beactive in a developmentally regulated manner, or at a basal level inother organs or tissues within the plant as well.

An inducible regulatory region is one that is capable of directly orindirectly activating transcription of one or more DNA sequences orgenes in response to an inducer. In the absence of an inducer the DNAsequences or genes will not be transcribed. Typically the proteinfactor, which binds specifically to an inducible regulatory region toactivate transcription, may be present in an inactive form which is thendirectly or indirectly converted to the active form by the inducer.However, the protein factor may also be absent. The inducer can be achemical agent such as a protein, metabolite, growth regulator,herbicide or phenolic compound or a physiological stress imposeddirectly by heat, cold, salt, or toxic elements or indirectly throughthe action of a pathogen or disease agent such as a virus. A plant cellcontaining an inducible regulatory region may be exposed to an inducerby externally applying the inducer to the cell or plant such as byspraying, watering, heating or similar methods. Inducible regulatoryelements may be derived from either plant or non-plant genes (e.g. Gatz,C. and Lenk, I. R. P., 1998; which is incorporated by reference).Examples, of potential inducible promoters include, but not limited to,teracycline-inducible promoter (Gatz, C., 1997; which is incorporated byreference), steroid inducible promoter (Aoyama, T. and Chua, N. H.,1997; which is incorporated by reference) and ethanol-inducible promoter(Salter, M. G., et al, 1998; Caddick, M. X. et al,1998; which areincorporated by reference) cytokinin inducible IB6 and CKI1 genes(Brandstatter, I. and Kieber, J. J., 1998; Kakimoto, T., 1996; which areincorporated by reference) and the auxin inducible element, DR5(Ulmasov, T., et al., 1997; which is incorporated by reference).

A constitutive regulatory region directs the expression of a genethroughout the various parts of a plant and continuously throughoutplant development. Examples of known constitutive regulatory elementsinclude promoters associated with the CaMV 35S transcript. (Odell etal., 1985), the rice actin 1 (Zhang et al, 1991), actin 2 (An et al.,1996), or tms 2 (U.S. Pat. No. 5,428,147, which is incorporated hereinby reference), and triosephosphate isomerase 1 (Xu et. al., 1994) genes,the maize ubiquitin 1 gene (Comejo et al, 1993), the Arabidopsisubiquitin 1 and 6 genes (Holtorf et al, 1995), and the tobaccotranslational initiation factor 4A gene (Mandel et al, 1995). The term“constitutive” as used herein does not necessarily indicate that a geneunder control of the constitutive regulatory region is expressed at thesame level in all cell types, but that the gene is expressed in a widerange of cell types even though variation in abundance is oftenobserved.

The regulatory regions of the first and second nucleotide sequencesdenoted above, may be the same or different. For example, which is notto be considered limiting in any manner, the regulatory elements of thefirst and second genetic constructs may both be constitutive. In anaspect of an embodiment, the first and second nucleotide sequences maybe maintained in the same plant. In an alternate embodiment the firstand second nucleotide sequences are maintained in separate plants, afirst and a second plant, respectively. The first nucleotide sequenceencoding a nucleic acid sequence of interest is expressed within thefirst plant. In the second embodiment, the second plant expresses thesecond nucleic acid sequence encoding the fusion protein capable ofregulating the expression of the nucleic acid sequence of interestwithin the first plant. Crossing of the first and second plants producesa progeny that expresses the fusion protein which regulates theexpression of the nucleic acid sequence of interest. In this manner theexpression of nucleic acid sequence of interest that is required tomaintain parent stocks may be retained within a parent plant but notexpressed in a progeny plant. Such a cross may produce sterileoffspring.

Alternatively, which is not to be considered limiting in any manner, thesecond regulatory element may be active before, during, or after thefirst regulatory element is active. Similarly, the first regulatoryelement may be active before, during, or after the second regulatoryelement is active. Other examples, which are not to be consideredlimiting, include the second regulatory element being an inducibleregulatory element that is activated by an external stimulus so thatregulation of gene expression may be controlled through the addition ofan inducer. The second regulatory element may also be active during aspecific developmental stage preceding, during, or following that of theactivity of the first regulatory element. In this way the expression ofthe nucleic acid sequence of interest may be repressed or activated asdesired within a plant.

By “nucleic acid sequence of interest”, “nucleotide sequence ofinterest” or “coding region of interest” it is meant any gene ornucleotide sequence that is to be expressed within a host organism. Sucha nucleotide sequence of interest may include, but is not limited to, agene whose product has an effect on plant growth or yield, for example aplant growth regulator such as an auxin or cytokinin and theiranalogues, or a nucleotide sequence of interest may comprise a herbicideor a pesticide resistance gene, which are well known within the art. Anucleic acid sequence of interest or a coding region of interest, mayencode an enzyme involved in the synthesis of, or in the regulation ofthe synthesis of, a product of interest, for example, but not limited toa protein, or an oil product. A nucleotide sequence of interest or acoding region of interest, may encode an industrial enzyme, proteinsupplement, nutraceutical, or a value-added product for feed, food, orboth feed and food use. Examples of such proteins include, but are notlimited to proteases, oxidases, phytases, chitinases, invertases,lipases, cellulases, xylanases, enzymes involved in oil biosynthesis,etc.

A nucleotide sequence of interest or a coding region of interest, mayalso encode a pharmaceutically active protein, for example growthfactors, growth regulators, antibodies, antigens, their derivativesuseful for immunization or vaccination and the like. Such proteinsinclude, but are not limited to, interleukins, insulin, G-CSF, GM-CSF,hPG-CSF, M-CSF or combinations thereof, interferons, for example,interferon-α, interferon-β, interferon-γ, blood clotting factors, forexample, Factor VIII, Factor IX, or tPA or combinations thereof. If thenucleic acid sequence of interest or a coding region of interest,encodes a product that is directly or indirectly toxic to the plant,then by using the method of the present invention, such toxicity may bereduced throughout the plant by selectively expressing the nucleic acidsequence of interest within a desired tissue or at a desired stage ofplant development.

A nucleotide sequence of interest or a coding region of interest, mayalso include a gene that encodes a protein involved in regulation oftranscription, for example DNA-binding proteins that act as enhancers orbasal transcription factors. Moreover, a nucleotide sequence of interestmay be comprised of a partial sequence or a chimeric sequence of any ofthe above genes, in a sense or antisense orientation.

It is also contemplated that a nucleic acid sequence of interest or acoding region of interest, may be involved in the expression of a geneexpression cascade, for example but not limited to a developmentalcascade. In this embodiment, the nucleic acid sequence of interest ispreferably associated with a gene that is involved at an early stagewithin the gene cascade, for example homeotic genes. Expression of anucleic acid sequence of interest, for example a repressor of homeoticgene expression, represses the expression of a homeotic gene. Expressionof the fusion protein that represses gene expression within the sameplant, either via crossing, induction, temporal or developmentalexpression of the regulatory region, as described herein, de-repressesthe expression of the homeotic gene thereby initiating a gene cascade.Conversely, using the methods described herein, expression of anintroduced (i.e. transgenic) homeotic gene may be activated in aselective manner, so that it is expressed outside of its normaldevelopmental or temporal expression pattern, thereby initiating acascade of developmental events. This may be achieved by targeting achromatin remodelling protein to a desired homeotic gene as describedherein.

Homeotic genes are well known to one of skill in the art, and includebut are not limited to, transcription factor proteins and associatedregulatory regions, for example controlling sequences that bind AP2domain containing transcription factors, for example but not limited to,APETALA2 (a regulator of meristem identity, floral organ specification,seedcoat development and floral homeotic gene expression; Jofuku et al.,1994), CCAAT box-binding transcription factors (e.g. LEC1; WO 98/37184;Lotan, T. et al., 1998), or the controlling factor associated withPICKLE, a gene that produces a thickened, primary root meristem (Ogas,J. et al.,1997).

A nucleic acid sequence of interest or a coding region of interest, mayalso be involved in the control of transgenes across generations, orproduction of F1 hybrid plants with seed characteristics that would beundesirable in the parental line or progeny, for example but not limitedto, oil seeds characterized as having reduced levels of sinapinebiosynthesis within the oil-free meal. In this case, a nucleic acidsequence of interest may be any enzyme involved in the synthesis of oneor more intermediates in sinipine biosynthesis. An example, which is tobe considered non-limiting, is caffeic o-methyltransferase (Acc#AAG51676), which is involved in ferulic acid biosynthesis. Otherexamples of genes of interest include genes that encode proteinsinvolved in fiber, or glucosinolate, biosynthesis, or a protein involvedin the biosynthesis of a phytotoxin. Phytotoxins may also be used forplant selection purposes. In this non-limiting example, a nucleic acidsequence of interest may encode a protein that is capable ofmetabolizing a compound from a non-toxic form to a toxic form therebyselectively removing plants that express the nucleic acid sequence ofinterest. The phytotoxic compound may be synthesized from endogenousprecursors that are metabolized by the nucleic acid sequence of interestinto a toxic form, for example plant growth regulators, or thephytotoxic compound may be synthesized from an exogenously appliedcompound that is only metabolized into a toxic compound in the presenceof the nucleic acid sequence of interest. For example, which is not tobe considered limiting, the nucleic acid sequence of interest maycomprise indole acetamide hydrolase (IAH), that converts exogenouslyapplied indole acetamide (IAM) or naphthaline acetemide (NAM), to indoleacetic acid (IAA), or naphthaline acetic acid (NAA), respectively.Over-synthesis of TAA or NAA is toxic to a plant, however, in theabsence of TAH, the applied IAM or NAM is non-toxic. Similarly, thenucleic acid sequence of interest may encode a protein involved inherbicide resistance, for example, but not limited to, phosphinothricinacetyl transferase, wherein, in the absence of the gene encoding thetransferase, application of phosphinothricin, the toxic compound(herbicide) results in plant death. Other nucleic acid sequence ofinterest that encode lethal or conditionally lethal products may befound in WO 00/37660 (which is incorporated herein by reference).

The nucleic acid sequence of interest, the nucleotide sequence ofinterest or a coding region of interest, may be expressed in suitableeukaryotic hosts which are transformed by the nucleotide sequences, ornucleic acid molecules, or genetic constructs, or vectors of the presentinvention. Examples of suitable hosts include, but are not limited to,insect hosts, mammalian hosts, yeasts and plants. Suitable plant hostsinclude, but are not limited to agricultural crops including canola,Brassica spp., maize, tobacco, alfalfa, rice, soybean, wheat, barley,sunflower, and cotton.

The one or more chimeric genetic constructs of the present invention canfurther comprise a 3′ untranslated region. A 3′ untranslated regionrefers to that portion of a gene comprising a DNA segment that containsa polyadenylation signal and any other regulatory signals capable ofeffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by effecting the addition of polyadenylic acidtracks to the 3′ end of the mRNA precursor. Polyadenylation signals arecommonly recognized by the presence of homology to the canonical form5′-AATAAA-3′ although variations are not uncommon. One or more of thechimeric genetic constructs of the present invention can also includefurther enhancers, either translation or transcription enhancers, as maybe required. These enhancer regions are well known to persons skilled inthe art, and can include the ATG initiation codon and adjacentsequences. The initiation codon must be in phase with the reading frameof the coding sequence to ensure translation of the entire sequence.

Examples of suitable 3′ regions are the 3′ transcribed non-translatedregions containing a polyadenylation signal of Agrobacterium tumorinducing (Ti) plasmid genes, such as the nopaline synthase (Nos gene)and plant genes such as the soybean storage protein genes and the smallsubunit of the ribulose-1,5-bisphosphate carboxylase (ssRUBISCO) gene.

To aid in identification of transformed plant cells, the constructs ofthis invention may be further manipulated to include selectable markers.Useful selectable markers in plants include enzymes which provide forresistance to chemicals such as an antibiotic for example, gentamycin,hygromycin, kanamycin, or herbicides such as phosphinothrycin,glyphosate, chlorosulfuron, and the like. Similarly, enzymes providingfor production of a compound identifiable by colour change such as GUS(β-glucuronidase), or luminescence, such as luciferase or GFP, areuseful.

Also considered part of this invention are transgenic eukaryotes, forexample but not limited to plants containing the chimeric gene constructof the present invention. However, it is to be understood that thechimeric gene constructs of the present invention may also be combinedwith nucleic acid sequence of interest for expression within a range ofeukaryotic hosts.

In instances where the eukaryotic host is a plant, methods ofregenerating whole plants from plant cells are also known in the art. Ingeneral, transformed plant cells are cultured in an appropriate medium,which may contain selective agents such as antibiotics, where selectablemarkers are used to facilitate identification of transformed plantcells. Once callus forms, shoot formation can be encouraged by employingthe appropriate plant hormones in accordance with known methods and theshoots transferred to rooting medium for regeneration of plants. Theplants may then be used to establish repetitive generations, either fromseeds or using vegetative propagation techniques. Transgenic plants canalso be generated without using tissue cultures (for example, Clough andBent, 1998).

The constructs of the present invention can be introduced into plantcells using Ti plasmids, Ri plasmids, plant virus vectors, direct DNAtransformation, micro-injection, electroporation, etc. For reviews ofsuch techniques see for example Weissbach and Weissbach, 1988; Geiersonand Corey, 1988; and Miki and Iyer, 1997; Clough and Bent, 1998). Thepresent invention further includes a suitable vector comprising thechimeric gene construct.

The DNA binding protein which is employed in the method of the presentinvention may be naturally produced in an organism other than a plant.For example, but not wishing to be considered limiting, a ROS repressoris encoded by a nucleotide sequence of bacterial origin and, as such thenucleotide sequence may be optimised, for example, by changing itscodons to favour plant codon usage, by attaching a nucleotide sequenceencoding a nuclear localisation signal (NLS), for example but notlimited to SV40 localization signal (see Robbins et al., 1991; Rizzo,P., et al., 1991; which are incorporated herein by reference) in orderto improve the efficiency of ROS transport to the plant nucleus tofacilitate the interaction with its respective operator, or bothoptimizing plant codon usage. Addition of an NLS to a fusion proteincomprising a binding domain, for example the ROS repressor bindingdomain, and a recruitment factor, may also ensure targeting of thefusion product to the nuclear compartment. Similar optimization may beperformed for other DNA binding proteins of non-plant source, however,such optimization may not always be required. Other possible nuclearlocalization signals that may be fused to a DNA binding protein includebut are not limited to those listed in Table 1:

TABLE 1 nuclear localization signals Nuclear Protein Organism NLS RefAGAMOUS A RienttnrqvtfcKRR (SEQ ID NO:13) 1 TGA-1A T RRlaqnreaaRKsRlRKK(SEQ ID NO:14) 2 TGA-1B T KKRaRlvrnresaqlsRqRKK (SEQ ID NO:15) 2 O2 NLSB M RKRKesnresaRRsRyRK (SEQ ID NO:16) 3 NIa V KKnqkhklkm-32aa-KRK (SEQID NO:17) 4 Nucleoplasmin X KRpaatkkagqaKKKKl (SEQ ID NO:18) 5 NO38 XKRiapdsaskvpRKKtR (SEQ ID NO:19) 5 N1/N2 X KRKteeesplKdKdaKK (SEQ IDNO:20) 5 Glucocorticoid receptor M,R RkclqagmnleaRKtKK (SEQ ID NO:21) 5a receptor H RKclqagmnleaRKtKK (SEQ ID NO:22) 5 β receptor HRKclqagmnleaRKtKK (SEQ ID NO:23) 5 Progesterone C,H,Ra RKccqagmvlggRKfKK(SEQ ID NO:24) 5 receptor Androgen H RKcyeagmtlgaRKlKK (SEQ ID NO:25) 5receptor p53 C RRcfevrvcacpgRdRK (SEQ ID NO:26) 5 ⁺A, Arabidopsis; X,Xenopus; M, mouse; R, rat; Ra, rabbit; H, human; C, chicken; T, tobacco;M, maize; V, potyvirus. References: 1. Yanovsky et al., 1990 2. van derKrol and Chua, 1991 3. Varagona et al., 1992 4. Carrington et al., 19915. Robbins et al., 1991

Incorporation of a nuclear localization signal into the fusion proteinof the present invention may facilitate migration of the fusion protein,into the nucleus. Without wishing to be bound by theory, reduced levelsof fusion proteins elsewhere within the cell may be important when theDNA binding portion of the fusion protein may bind analogue operatorsequences within other organelles, for example within the mitochondrionor chloroplast. Furthermore, the use of a nuclear localization signalmay permit the use of a less active promoter or regulatory region todrive the expression of the fusion protein while ensuring that theconcentration of the expressed protein remains at a desired level withinthe nucleus, and that the concentration of the protein is reducedelsewhere in the cell.

Referring now to FIGS. 2A-C, there is shown aspects of an embodiment ofthe method of the present invention. Shown in FIG. 2A are two constructswhich have been introduced within a plant cell. The constructs comprise:

-   -   1) a first nucleotide sequence (10) comprising,        -   a) a nucleic acid sequence of interest (20) operatively            linked to a first regulatory region (30);        -   b) an operator sequence (40) capable of binding a fusion            protein (85, FIG. 2B), and;    -   2) a second nucleotide sequence (60) comprising a second        regulatory region (70) in operative association with a        nucleotide sequence (80) encoding a fusion protein (85).        The fusion protein (FIG. 2B; 85) encoded by nucleotide        sequence (80) comprises    -   a) a DNA binding protein (100), or a portion of a DNA binding        protein capable of binding the operator sequence (40, FIG. 2A),        and;    -   b) a recruitment factor protein (110), or a portion of a        recruitment factor protein capable of binding a chromatin        remodelling protein (120), for example but not limited to        histone deacetylase, HDAC.        In the example shown in FIG. 2A-C, the operator sequence (40) is        shown as being upstream from the regulatory region (30),        however, the operator sequence may also be positioned downstream        from the regulatory region (40), for example between the        regulatory region (40) and the nucleic acid sequence of interest        (20; see for example the constructs in FIG. 5A-D), within the        coding region of the nucleic acid sequence of interest (20), or        downstream of the nucleic acid sequence of interest (20).

Referring now to FIG. 2C, but without wishing to be bound by theory,transcription and translation of nucleotide sequence (60; FIG. 2A)produces fusion protein (80; FIG. 2B) which is capable of bindingoperator sequence (40; FIG. 2A) and for example, histone deacetylase(120). Dual binding of histone deacetylase (120) to fusion protein (85)and fusion protein (85) to operator sequence (40) facilitates enzymaticdeacetylation of histones (via bound histone deacetylase) in proximityof the nucleic acid sequence of interest (20) thereby causing repressionof the nucleic acid sequence of interest (20).

The first (10) and second (60) nucleotide sequences may be placed withinthe same or within different vectors, genetic constructs, or nucleicacid molecules. Preferably, the first nucleotide sequence and the secondnucleotide sequence are chromosomally integrated into a plant or plantcell. The two nucleotide sequences may be integrated into two differentgenetic loci of a plant or plant cell, or the two nucleotide sequencesmay be integrated into a singular genetic locus of a plant or plantcell. However, the second nucleotide sequence may be integrated into theDNA of the plant or it may be present as an extra-chromosomal element,for example, but not wishing to be limiting a plasmid. Furthermore, thefirst and second regulatory regions may be the same or different, andmaybe active in a constitutive, temporal, developmental or induciblemanner.

Referring now to FIGS. 3A-C, there is shown aspects of an alternateembodiment of the method of the present invention. Shown in FIG. 3A aretwo constructs which have been introduced into a plant cell. Theconstructs comprise:

-   -   1) a first nucleotide sequence (10) comprising,        -   a) a nucleic acid sequence of interest (20) operatively            linked to a regulatory region (30),        -   b) an operator sequence (40) capable of binding a fusion            protein (85, FIG. 3B), and;    -   2) a second nucleotide sequence (60) comprising a regulatory        region (70) in operative association with a nucleotide        sequence (80) encoding a fusion protein (85).        The fusion protein (85) encoded by nucleotide sequence (80)        comprises,    -   a) a DNA binding protein (100), or a portion of a DNA binding        protein capable of binding the operator sequence (40), and;    -   b) a recruitment factor protein (110), or a portion of a        recruitment factor protein capable of binding a chromatin        remodelling protein, for example but not limited, to free        histone acetyltransferase (HAT) (120).        In the example shown in FIG. 3A-C, the operator sequence (40) is        shown as being upstream from the regulatory region (30),        however, the operator sequence may also be positioned downstream        from the regulatory region (40), for example between the        regulatory region (40) and the nucleic acid sequence of interest        (20; see for example the constructs in FIG. 5A-D), within the        coding region of the nucleic acid sequence of interest (20), or        downstream of the nucleic acid sequence of interest (20).

Referring now to FIG. 3C, but without wishing to be bound by theory,transcription and translation of nucleotide sequence (80; FIG. 3A)produces fusion protein (85; FIG. 3B) which is capable of bindingoperator sequence (40; FIG. 3A) and free histone acetyltransferase(120). Dual binding of histone acetyltransferase (120) to fusion protein(85) and fusion protein (85) to operator sequence (40) facilitatesenzymatic acetylation of histones (via bound histone acetyltransferase)in proximity of the nucleic acid sequence of interest (20) therebycausing an increase in the transcription of the nucleic acid sequence ofinterest (20).

The present invention also relates to a method of enhancing theexpression of a nucleic acid sequence of interest or enhancing thetranscription of one or more selected nucleotide sequences bytransforming a plant with one or more constructs comprising:

-   -   1) a first nucleotide sequence comprising,        -   a) a nucleic acid sequence of interest operatively linked to            a regulatory region, and;        -   b) an operator sequence that interacts with a fusion            protein;    -   2) a second nucleotide sequence comprising a regulatory region        in operative association with a nucleotide sequence encoding a        fusion protein comprising,        -   a) a DNA binding protein, or a portion of a DNA binding            protein capable of binding the operator sequence, and;        -   b) a histone acetyltransferase (HAT) protein, or portion of            a histone acetyltransferase protein which is capable of            increasing histone acetylation;

and wherein binding of the fusion protein to the operator sequenceincreases histone acetylation in the proximity of the nucleic acidsequence of interest within the first nucleotide sequence therebyincreasing the transcription of the nucleic acid sequence of interest.

These first and second nucleotide sequences may be placed within thesame or within different vectors, genetic constructs, or nucleic acidmolecules. Preferably, the first nucleotide sequence and the secondnucleotide sequence are chromosomally integrated into a plant or plantcell. The two nucleotide sequences may be integrated into two differentgenetic loci of a plant or plant cell, or the two nucleotide sequencesmay be integrated into a singular genetic locus of a plant or plantcell. However, the second nucleotide sequence may be integrated into theDNA of the plant or it may be present as an extra-chromosomal element,for example, but not wishing to be limiting a plasmid, or transientlyexpressed, for example when using viral vectors, bioloistics fortransformation.

Preferably, the operator sequence is located in a nucleotide region thatdoes not sterically hinder binding of transcription factors to theregulatory region, binding of the RNA polymerase to the nucleic acidsequence of interest, or migration of the polymerase along the DNA ofthe first nucleotide sequence, nucleic acid sequence of interest orboth.

Referring now to FIGS. 4A-C, there is shown aspects of an embodiment ofthe method of the present invention. Shown in FIG. 4A are two constructswhich have been introduced within a plant cell. The constructs comprise:

-   -   1) a first nucleotide sequence (10) comprising,        -   a) a nucleic acid sequence of interest (20) operatively            linked to a regulatory region (30),        -   b) an operator sequence (40) capable of binding a fusion            protein (85), and;    -   2) a second nucleotide sequence (60) comprising a regulatory        region (70) in operative association with a nucleotide        sequence (80) encoding a fusion protein (85).        The fusion protein (85) encoded by nucleotide sequence (80)        comprises    -   a) a DNA binding protein (100), or a portion of a DNA binding        protein capable of binding the operator sequence (40), and;    -   b) a histone acetyltransferase protein (130), or a portion of a        histone acetyltransferase protein.

Referring now to FIG. 4C, but without wishing to be bound by theory,transcription and translation of nucleotide sequence (80; FIG. 4A)produces fusion protein (85; FIG. 4B) which comprises an active HATprotein (130), or portion thereof. Binding of the fusion protein (85) tothe operator sequence facilitates enzymatic acetylation of histones inproximity to the nucleic acid sequence of interest (20) therebyenhancing the expression of a nucleic acid sequence of interest.

In the example shown in FIG. 4 A-C, the operator sequence (40) is shownas being upstream from the regulatory region (30), however, the operatorsequence may also be positioned downstream from the regulatory region(40), for example between the regulatory region (40) and the nucleicacid sequence of interest (20; see for example the constructs in FIG.5A-D), within the coding region of the nucleic acid sequence of interest(20), or downstream of the nucleic acid sequence of interest (20).

Also contemplated by the present invention is the control of geneexpression accomplished through combinations of activator, effector andgene of interest constructs as outlined in FIGS. 29A and B (see Example6). With reference to FIG. 29A, the expression of a gene of interest(reporter) is regulated using three constructs:

-   -   a reporter construct (or gene of interest construct),    -   an activator construct and    -   an effector construct.        The gene of interest construct includes a gene of interest, for        example but not limited to a reporter gene (e.g. the lacZ gene),        in operative association with a regulatory element and an        operator sequence.

The activator construct comprises a nucleic acid sequence encoding arecruitment factor protein, or a portion thereof, capable of binding achromatin remodelling protein, fused with a nucleotide sequence encodinga DNA binding protein, or a fragment thereof. The recruitment factorprotein may be, for example but not limited to BnSCL1, bnKCP1 or anactive fragment thereof; the DNA binding protein could be, for examplebut not limited to VP16 or GAL4 DNA Binding domain. In this case theactivator construct produces a VP16-bNSCL1 fusion protein.

The effector plasmid includes a nucleic acid sequence encoding achromatin remodelling factor, for example but not limited to HDA19,operatively associated with a regulatory element and a nucleic acidsequence encoding a nuclear localisation signal. The constructs areexpressed in eukaryotes, for example plant, animal or yeast.

When the activator construct is co-expressed with the gene of interest(reporter) construct, the DNA binding sequence binds the operatorsequence of the gene of interest construct. This results in modificationin the expression of the gene of interest due to interaction of theactivator protein within the transcriptional machinery. In this example,the activator protein is fused to a recruitment factor protein, and theVP16-BnSCL1 fusion protein binds the Tet operator sequence of the geneactivator construct resulting in increased expression of the gene ofinterest.

Co-expression of the effector construct, inconjucntion with the gene ofinterst and activator constructs, results in synthesis of a chromatinremodelling factor, in this case HAD19, which associates with therecruitment factor protein, BnSCL1. Association of HDAC with theconstruct expressing the gene of interst, reduces expression of the geneof interest.

In a second aspect, the expression of a gene of interest is regulatedusing two constructs: a gene of interest (reporter)+activator and aneffector construct as shown in FIG. 29B. Expression of thereporter+activator construct results in an increased expression of thegene of interest due to binding of the activator portion of theconstruct to the operator sequence of the gene of interest construct.This association may be inhibited in the presence of tetracycline. As inthe case outlined with reference to FIG. 29A, above, co-expression ofthe effector construct results in reduced expression of the gene ofinterest due to association of HDAC to the activator-recruitment factorfusion protein (VP16-BnSCL1 fusion)

The present invention also provides for a method to regulate expressionof a nucleic acid sequence of interest, wherein the nucleic acidsequence of interest comprises an endogenous sequence. In thisembodiment, a nucleotide sequence comprising a regulatory region inoperative association with a nucleotide sequence encoding a recruitmentfactor, or a portion thereof, that is known to interact with a factorthat binds the nucleic acid sequence of interest, is expressed in thehost. The recruitment factor protein, or a portion thereof is capable ofbinding a chromatin remodelling protein, for example but not limited,HDAC or HAT, and the recruitment factors also interacts with endogenousfactors that bind the nucleotide sequence of interest (e.g.transcription factors). In this manner, expression of the recruitmentfactor in a temporal, tissue specific, or induced manner will result inthe expression of the recruitment factor that binds the chromatinremodelling factor and transcription factor resulting in modulation ofexpression of the nucleic acid sequence of interest. A non-limitingexample of this embodiment includes the expression of bnKCP1 and itsinteraction with HDAC and transcription factors ERF, SEBF or CBF.

Therefore, the present invention provides a method to regulateexpression of an endogenous nucleic acid sequence of interest in a plantcomprising:

-   -   i) introducing into the plant a nucleotide sequence comprising,        a regulatory region, operatively linked with a nucleotide        sequence encoding a recruitment factor protein, the recruitment        factor protein capable of binding an endogenous DNA binding        protein, the endogenous DNA binding protein characterized in        binding a segment of a DNA sequence of the endogenous nucleotide        sequence of interest, and;    -   ii) growing the plant, wherein expression of the nucleotide        sequence produces the recruitment factor thereby regulating        expression of the endogenous nucleic acid sequence of interest.

An alternate embodiment of the present invention includes a method toregulate expression of an endogenous nucleic acid sequence of interest.In this example, a DNA binding protein, or a portion thereof, known tointeract with the DNA of an endogenous nucleic acid sequence of interestis fused to a chromatin remodelling factor. Expression of the fusionprotein permits the recruitment factor portion of the fusion protein tointeract or bind with a chromatin remodelling, for example but notlimited to HDAC or HAT, and the DNA binding portion of the fusionprotein binds the nucleotide sequence of interest. In this manner,expression of the fusion protein in a temporal, tissue specific, orinduced manner will result in the expression of a recruitment factorthat binds a chromatin remodelling factor and the DNA of a nucleic acidsequence of interest, resulting in modulation of expression of theendogenous nucleic acid sequence of interest. Examples of DNA bindingproteins, or portions thereof, that bind endogenous nucleic acidsequences of interest, which are not to be considered limiting, includeERF, SEBF or CBF. A non-limiting example of a recruitment factor isbnKCP1 or BnSCL1.

Therefore, the present invention also provides a method to regulateexpression of an endogenous nucleic acid sequence of interest in a plantcomprising:

-   -   i) introducing into the plant a nucleotide sequence comprising,        a regulatory region, operatively linked with a nucleotide        sequence encoding a fusion protein, the fusion protein        comprising,        -   a) a DNA binding protein, or a portion thereof, capable of            binding a segment of a DNA sequence of the endogenous            nucleotide sequence of interest, and;        -   b) a recruitment factor protein, or a portion thereof,            capable of binding a chromatin remodelling protein; and    -   ii) growing the plant, wherein expression of the nucleotide        sequence produces the fusion protein that regulates expression        of the endogenous nucleic acid sequence of interest.

Also contemplated by the present invention is a method of increasingcold tolerance in a plant. The method comprises providing a plant havinga nucleotide sequence of interest operatively linked to a firstregulatory region; the nucleotide sequence of interest encodes bnKCP1,or a fragment thereof. The plant is maintained under conditions wherebnKCP1 is expressed. In this manner, the plant expressing bnKCP1 ispreconditioned for cold adaptation and exhibits increased coldtolerance.

By the term cold in the context of cold tolerance, it is meant atemperature in the range of about −10° C. to about 10° C. An example ofcold temperature, without wishing to be limiting, is a temperature inthe range of about −8° C. to about 8° C.; a further example is atemperature of about −10 to about −1° C.

Sequences of the present invention are listed in Table 2.

TABLE 2 SEQ ID NO:1 aa seq of wild-type ROS (A. tumefaciens) FIG. 1A(WT-ROS) SEQ ID NO:2 Nucl seq synthetic ROS optimized for plant, withNLS FIG. 1B SEQ ID NO:3 Consensus nucl seq of composite ROS FIG. 1C SEQID NO:4 aa seq of synthetic ROS FIG. 1A, 1C SEQ ID NO:5 ROS bindingsequence FIG. 1E SEQ ID NO:6 aa seq of NLS (PKKKRKV) SEQ ID NO:7 ROSoperator sequence SEQ ID NO:8 IPT gene operator sequence SEQ ID NO:9Operator sequence binding to ERF SEQ ID NO:10 Operator sequence bindingto SEBF SEQ ID NO:11 Operator sequence binding to CBF SEQ ID NO:12Operator sequence binding to CBF SEQ ID NO:13 NLS of AGAMOUS proteinTable 1, page 30 SEQ ID NO:14 NLS of TGA-1A protein Table 1, page 30 SEQID NO:15 NLS of TGA-1B protein Table 1, page 30 SEQ ID NO:16 NLS of O2NLS B protein Table 1, page 30 SEQ ID NO:17 NLS of NIa protein Table 1,page 30 SEQ ID NO:18 NLS of nucleoplasmin protein Table 1, page 30 SEQID NO:19 NLS of NO38 protein Table 1, page 30 SEQ ID NO:20 NLS of N1/N2protein Table 1, page 30 SEQ ID NO:21 NLS of Glucocorticoid receptorTable 1, page 30 SEQ ID NO:22 NLS of Glucocorticoid a receptor Table 1,page 30 SEQ ID NO:23 NLS of Glucocorticoid b receptor Table 1, page 30SEQ ID NO:24 NLS of Progesterone receptor Table 1, page 30 SEQ ID NO:25NLS of Androgen receptor Table 1, page 30 SEQ ID NO:26 NLS of p53protein Table 1, page 30 SEQ ID NO:27 VirC/VirD operator seq FIG. 1D SEQID NO:28 ROS-OPDS, p74-315 SEQ ID NO:29 ROS-OPDA, p74-315 SEQ ID NO:30ROS-OPUS, p74-316 SEQ ID NO:31 ROS-OPUA, p74-316 SEQ ID NO:32 ROS-OPPS,p74-309 SEQ ID NO:33 ROS-OPPA, p74-309 SEQ ID NO:34 ROS-OP1, p74-508 SEQID NO:35 ROS-OP2, p74-508 SEQ ID NO:36 tms2 promoter sense primer,p74-508 SEQ ID NO:37 tms2 promoter anti-sense primer, p74-508 SEQ IDNO:38 Actin2 promoter sense primer, p74-501 SEQ ID NO:39 Actin2 promoteranti-sense primer, p74-501 SEQ ID NO:40 p74-315 seq from EcoRV to ATG ofGUS SEQ ID NO:41 p74-316 seq from EcoRV to ATG of GUS SEQ ID NO:42p74-309 seq from EcoRV to ATG of GUS SEQ ID NO:43 p74-118 seq from EcoRVto ATG of GUS SEQ ID NO:44 Forward primer for HDA19 A. thaliana,pDBLeu-HDA19 SEQ ID NO:45 Reverse primer for HDA19 A. thaliana,pDBLeu-HDA19 SEQ ID NO:46 Forward primer for Gcn5 Arabidopsis, GST-Gcn5SEQ ID NO:47 Reverse primer for Gcn5 Arabidopsis, GST-Gcn5 SEQ ID NO:48Reverse primer for HDA19, GST-HDA19 SEQ ID NO:49 Forward primer forbnKCP1, 1-80, 1-160 (generation of mutants) SEQ ID NO:50 Reverse primerfor bnKCP1 1-160 (generation of mutants) SEQ ID NO:51 Reverse primer forbnKCP1 1-80 (generation of mutants) SEQ ID NO:52 Reverse primer forbnKCP1 (generation of mutants) SEQ ID NO:53 Forward primer for bnKCP1,1-80 and 1-160 (in vivo assay and transactivation assay) SEQ ID NO:54Reverse primer for bnKCP1 (in vivo assay and transactivation assay) and81-215 (transactivation assay) SEQ ID NO:55 Reverse primer for bnKCP11-160 (in vivo assay and transactivation assay) SEQ ID NO:56 Reverseprimer for bnKCP1 1-80 (in vivo assay and transactivation assay) SEQ IDNO:57 Forward primer for bnKCP1G188 SEQ ID NO:58 Reverse primer forbnKCP1G188 SEQ ID NO:59 Forward primer for bnKCP1 81-215(transactivation assay) SEQ ID NO:60 Forward primer for entire codingregion of bnKCP1 SEQ ID NO:61 Reverse primer for entire coding region ofbnKCP1 SEQ ID NO:62 pat7 NLS (PLNKKRR) SEQ ID NO:63 aa seq of ROSR (ROSrepressor) FIG. 1A SEQ ID NO:64 aa seq of ROSAR (ROS repressor) FIG. 1ASEQ ID NO:65 aa seq of MucR (ROS repressor) FIG. 1A SEQ ID NO:66VirC/VirD DNA binding site seq (1) FIG. 1D SEQ ID NO:67 VirC/VirD DNAbinding site seq (2) FIG. 1D SEQ ID NO:68 ipt DNA binding site seq (1)FIG. 1D SEQ ID NO:69 ipt DNA binding site seq (2) FIG. 1D SEQ ID NO:70Consensus DNA binding site seq FIG. 1D SEQ ID NO:71 bnKCP aa seq FIG.10A SEQ ID NO:72 atKCP aa seq FIG. 10A SEQ ID NO:73 atKCL1 aa seq FIG.10A SEQ ID NO:74 atKCL2 aa seq FIG. 10A SEQ ID NO:75 bnKCP aa seq FIG.10B SEQ ID NO:76 ATF-1 aa seq FIG. 10B SEQ ID NO:77 hyCREB aa seq FIG.10B SEQ ID NO:78 CREB aa seq FIG. 10B SEQ ID NO:79 CREM aa seq FIG. 10BSEQ ID NO:80 cCREM aa seq FIG. 10B SEQ ID NO:81 aa seq of BnSCL1 FIG. 20SEQ ID NO:82 aa seq of atSCL15 FIG. 20 SEQ ID NO:83 aa seq of 1sSCR FIG.20 SEQ ID NO:84 BnSCL1 sense primer SEQ ID NO:85 BnSCL1 anti-senseprimer SEQ ID NO:86 BnIAA1 sense primer SEQ ID NO:87 BnIAA1 anti-senseprimer SEQ ID NO:88 BnIAA12 sense primer SEQ ID NO:89 BnIAA12 anti-senseprimer SEQ ID NO:90 Forward primer for BnSCL1, BnSCL1¹⁻³⁵⁸, BnSCL1¹⁻²⁶¹,BnSCL1¹⁻²¹⁷ and BnSCL1¹⁻¹⁴⁵ for pET-28b vector SEQ ID NO:91 Reverseprimer for BnSCL1 for pET-28b vector SEQ ID NO:92 Reverse primer forBnSCL1¹⁻³⁵⁸ for pET-28b vector SEQ ID NO:93 Reverse primer forBnSCL1¹⁻²⁶¹ for pET-28b vector SEQ ID NO:94 Reverse primer forBnSCL1¹⁻²¹⁷ for pET-28b vector SEQ ID NO:95 Reverse primer forBnSCL1¹⁻¹⁴⁵ for pET-28b vector SEQ ID NO:96 Forward primer for BnSCL1,BnSCL1¹⁻³⁵⁸, BnSCL1¹⁻²⁶¹, BnSCL1¹⁻²¹⁷ and BnSCL1¹⁻¹⁴⁵ for pPC86 vectorSEQ ID NO:97 Forward primer for BnSCL1¹⁴⁶⁻³⁵⁸ for PC86 vector SEQ IDNO:98 Forward primer for BnSCL1²¹⁸⁻⁴³⁴ for PC86 vector SEQ ID NO:99Reverse primer for BnSCL1 and BnSCL1²¹⁸⁻⁴³⁴ for PC86 vector SEQ IDNO:100 Reverse primer for BnSCL1¹⁻³⁵⁸ for PC86 vector SEQ ID NO:101Reverse primer for BnSCL1¹⁻²⁶¹ for PC86 vector SEQ ID NO:102 Reverseprimer for BnSCL1¹⁻²¹⁷ for PC86 vector SEQ ID NO:103 Reverse primer forBnSCL1¹⁻¹⁴⁵ for PC86 vector SEQ ID NO:104 aa seq of LXXLLmotif(¹⁴⁸LGSLL¹⁵²)

The above description is not intended to limit the claimed invention inany manner, furthermore, the discussed combination of features might notbe absolutely necessary for the inventive solution.

The present invention will be further illustrated in the followingexamples. However it is to be understood that these examples are forillustrative purposes only, and should not be used to limit the scope ofthe present invention in any manner.

EXAMPLES

Materials and Methods

Plant Material

Wild type Arabidopsis thaliana, ecotype Columbia, seeds were germinatedon RediEarth (W. R. Grace & Co.) soil in pots covered with windowscreens under green house conditions (˜25° C., 16 hr light). Emergingbolts were cut back to encourage further bolting. Plants were used fortransformation once multiple secondary bolts had been generated.

Plant Transformation

Plant transformation was carried out according to the floral dipprocedure described in Clough and Bent (1998). Essentially,Agrobacterium tumefaciens transformed with the construct of interest(using standard methods as known in the art) was grown overnight in a100 ml Luria-Bertani Broth (10 g/L NaCl, 10 g/L tryptone, 5 g/L yeastextract) containing 50 μg/ml kanamycin. The cell suspension culture wascentrifuged at 3000×g for 15 min. The pellet was resuspended in 1 L ofthe transformation buffer (sucrose (5%), Silwet L77 (0.05%)(LovelandIndustries). The above-ground parts of the Arabidopsis plants weredipped into the Agrobacterium suspension for ˜1 min and the plants werethen transferred to the greenhouse. The entire transformation processwas repeated twice more at two day intervals. Plants were grown tomaturity and seeds collected. To select for transformants, seeds weresurface sterilized by washing in 0.05% Tween 20 for 5 minutes, with 95%ethanol for 5 min, and then with a solution containing sodiumhypochlorite (1.575%) and Tween 20 (0.05%) for 10 min followed by 5washings in sterile water. Sterile seeds were plated onto either PeteLite medium (20-20-20 Peter's Professional Pete Lite fertilizer (Scott)(0.762 g/l), agar (0.7%), kanamycin (50 μg/ml), pH 5.5) or MS medium (MSsalts (0.5×)(Sigma), B5 vitamins (1×), agar (0.7%), kanamycin (50 μg/ml)pH 5.7). Plates were incubated at 20° C., 16 hr light/8 hr dark in agrowth room. After approximately two weeks, seedlings possessing greenprimary leaves were transferred to soil for further screening andanalysis:

Example 1 Optimization of ROS Protein Coding Region

The ros nucleotide sequence is derived from Agrobacterium tumefaciens(SEQ ID NO:1; FIG. 1A). Analysis of the protein coding region of the rosnucleotide sequence indicates that the codon usage may be altered tobetter conform to plant translational machinery. The protein codingregion of the ros nucleotide sequence was therefore modified to optimizeexpression in plants (SEQ ID NO:2; FIG. 1B). The nucleic acid sequenceof the ROS repressor was examined and the coding region modified tooptimize for expression of the gene in plants, using a procedure similarto that outlined by Sardana et al. (1996). A table of codon usage fromhighly expressed genes of dicotyledonous plants was compiled using thedata of Murray et al. (1989). The ros nucleotide sequence was alsomodified (SEQ ID NO:2; FIG. 1B) to ensure localization of the ROSrepressor to the nucleus of plant cells, by adding a SV40 nuclearlocalization signal (Rizzo, P. et al., 1999; The nuclear localizationsignal resides at amino acid positions 126-132; accession numberAAF28270).

The ros gene is cloned from Agrobacterium tumefaciens by PCR. Thenucleotide sequence encoding the ROS protein is expressed in, andpurified from, E. coli, and the ROS protein used to generate an anti-ROSantiserum in rabbits using standard methods (Sambrook et al.).

Example 2 Constructs Placing a Nucleic Acid Sequence of Interest UnderTranscriptional Control of Regulatory Regions that have been Modified toContain ROS Operator Sites, and Preparation of Reporter Lines

p74-315: Construct for The Expression of GUS Gene Driven by a CaMV 35SPromoter Containing a ROS Operator Downstream of TATA Box (FIG. 5(A)).

The BamHI-EcoRV fragment of CaMV 35S promoter in pBI121 is cut out andreplaced with a similar synthesized DNA fragment in which the 25 bpimmediately downstream of the TATA box were replaced with the ROSoperator sequence:

TATATTTCAATTTTATTGTAATATA. (SEQ ID NO: 7)Two complementary oligos, ROS-OPDS (SEQ ID NO:28) and ROS-OPDA (SEQ IDNO:29), with built-in BamHI-EcoRV ends, and spanning the BamHI-EcoRVregion of CaMV35S, in which the 25 bp immediately downstream of the TATAbox are replaced with the ROS operator sequence (SEQ ID NO: 7), areannealed together and then ligated into the BamHI-EcoRV sites ofCaMV35S.

(SEQ ID NO:28) ROS-OPDS: 5′-ATC TCC ACT GAC GTA AGG GAT GAC GCA CAA TCCCAC TAT CCT TCG CAA GAC CCT TCC TCT ATA TAA TAT ATT TCA ATT TTA TTG TAATAT AAC ACG GGG GAC TCT AGA G- 3′ (SEQ ID NO:29) ROS-OPDA: 5′-G ATC CTCTAG AGT CCC CCG TGT TAT ATT ACA ATA AAA TTG AAA TAT ATT ATA TAG AGG AAGGGT CTT GCG AAG GAT AGT GGG ATT GTG CGT CAT CCC TTA CGT CAG TGG AGA T-3′The p74-315 sequence from the EcoRV site (GAT ATC) to the first codon(ATG) of GUS is shown below (TATA box—lower case in bold; the syntheticROS sequence—bold caps; a transcription start site—ACA, bold italics;BamHI site—GGA TCC; and the first of GUS, ATG, in italics; are alsoindicated):

(SEQ ID NO:40) 5′-GAT ATC TCC ACT GAC GTA AGG GAT GAC GCA CAA TCC CACTAT CCT TCG CAA GAC CCT TCC TCt ata taA TAT ATT TCA ATT TTA TTG TAA TATA

CG GGG GAC TCT AGA GGA TCC CCG GGT GGT CAG TCC CTT ATG-3′p74-316: Construct for The Expression of GUS Driven by a CaMV 35SPromoter Containing a ROS Operator Upstream of TATA Box (FIG. 5(B)).

The BamHI-EcoRV fragment of CaMV 35S promoter in pBI121 is cut out andreplaced with a similar synthesized DNA fragment in which the 25 bpimmediately upstream of the TATA box are replaced with the ROS operatorsequence (SEQ ID NO: 7). Two complementary oligos, ROS-OPUS (SEQ IDNO:30) and ROS-OPUA (SEQ ID NO:31), with built-in BamHI-EcoRV ends, andspanning the BamHI-EcoRV region of CaMV35S, in which the 25 bpimmediately upstream of the TATA box were replaced with a ROS operatorsequence (SEQ ID NO: 7), are annealed together and then ligated into theBamHI-EcoRV sites of CaMV35S.

(SEQ ID NO:30) ROS-OPUS: 5′-ATC TCC ACT GAC GTA AGG GAT GAC GCA CAA TCTATA TTT CAA TTT TAT TGT AAT ATA CTA TAT AAG GAA GTT CAT TTC ATT TGG AGAGAA CAC GGG GGA CTC TAG AG-3′ (SEQ ID NO:31) ROS-OPUA: 5′-G ATC CTC TAGAGT CCC CCG TGT TCT CTC CAA ATG AAA TGA ACT TCC TTA TAT AGT ATA TTA CAATAA AAT TGA AAT ATA GAT TGT GCG TCA TCC CTT ACG TCA GTG GAG AT-3′The p74-316 sequence from the EcoRV site (GAT ATC) to the first codon(ATG) of GUS is shown below (TATA box—lower case in bold; the syntheticROS sequence—bold caps; a transcription start site—ACA, bold italics;BamHI site—GGA TCC; the first codon of GUS, ATG-italics, are alsoindicated):

(SEQ ID NO:41) 5′-GAT ATC TCC ACT GAC GTA AGG GAT GAC GCA CAA TCT ATATTT CAA TTT TAT TGT AAT ATA Cta tat aAG GAA GTT CAT TTC ATT TGG AGA GA

C GGG GGA CTC TAG AGG ATC CCC GGG TGG TCA GTC CCT TAT G-3′p74-309: Construct for The Expression of GUS Driven by a CaMV 35SPromoter Containing ROS Operators Upstream and Downstream of TATA Box(FIG. 5(C)).

The BamHI-EcoRV fragment of CaMV 35S promoter in pBI121 is cut out andreplaced with a similar synthesized DNA fragment in which the 25 bpimmediately upstream and downstream of the TATA box were replaced withtwo ROS operator sequences (SEQ ID NO: 7). Two complementary oligos,ROS-OPPS (SEQ ID NO:32) and ROS-OPPA (SEQ ID NO:33), with built-inBamHI-EcoRV ends, and spanning the BamHI-EcoRV region of CaMV35S, inwhich the 25 bp immediately upstream and downstream of the TATA box arereplaced with two ROS operator sequences, each comprising the sequenceof SEQ ID NO: 7 (in italics, below), are annealed together and ligatedinto the BamHI-EcoRV sites of CaMV35S.

(SEQ ID NO:32) ROS-OPPS: 5′-ATC TCC ACT GAC GTA AGG GAT GAC GCA CAA TCTATA TTT CAA TTT TAT TGT AAT ATA CTA TAT AAT ATA TTT CAA TTT TAT TGT AATATA ACA CGG GGG ACT CTA GAG-3′ (SEQ ID NO:33) ROS-OPPA: 5′-G ATC CTC TAGAGT CCC CCG TGT TAT ATT ACA ATA AAA TTG AAA TAT ATT ATA TAG TAT ATT ACAATA AAA TTG AAA TAT AGA TTG TGC GTC ATC CCT TAC GTC AGT GGA GAT-3′The p74-309 sequence from the EcoRV site (GAT ATC) to the first codon(ATG) of GUS is shown below (TATA box—lower case in bold; two syntheticROS sequence—bold caps; a transcription start site—ACA, bold italics;BamHI site—GGA TCC; the first codon of GUS, ATG-italics, are alsoindicated):

(SEQ ID NO:42) 5′-GAT ATC TCC ACT GAC GTA AGG GAT GAC GCA CAA TCT ATATTT CAA TTT TAT TGT AAT ATA Cta tat aAT ATA TTT CAA TTT TAT TGT AAT ATA

 CGG GGG ACT CTA GAG GAT CCC CGG GTG GTC AGT CCC TTA TG-3′p74-118 Construct for The Expression of GUS Driven by a CaMV 35SPromoter Containing three ROS Operators Downstream of TATA Box (FIG.5(D)).

The BamHI-EcoRV fragment of CaMV 35S promoter in pBI121 is cut out andreplaced with a similar synthesized DNA fragment in which a regiondownstream of the TATA box was replaced with three ROS operatorsequences (SEQ ID NO:43). The first of the three synthetic ROS operatorsequences is positioned immediately of the TAT box, the other two ROSoperator sequence are located downstream of the transcriptional startsite (ACA). Two complementary oligos with built-in BamHI-EcoRV ends wereprepared as describe above for the other constructs were annealedtogether and ligated into the BamHI-EcoRV sites of CaMV35S.

The p74-118 sequence from the EcoRV site (GAT ATC) to the first codon(ATG) of GUS is shown below (TATA box—lower case in bold; threesynthetic ROS sequence—bold caps; a transcription start site—ACA, bolditalics; BamHI site—GGA TCC; the first codon of GUS, ATG-italics, arealso indicated):

(SEQ ID NO:43) 5′-GAT ATC TCC ACT GAC GTA AGG GAT GAC GCA CAA TCC CACTAT CCT TCG CAA GAC CCT TCC TCt ata taA TAT ATT TCA ATT TTA TTG TAA TATA

 

CG GGG GAC TCT AGA GGA TCC TAT ATT TCA ATT TTA TTG TAA TAT AGC TAT ATTTCA ATT TTA TTG TAA TAT AAT CGA TTT CGA ACC CGG GGT ACC GAA TTC CTC GAGTCT AGA GGA TCC CCG GGT GGT CAG TCC CTT ATG-3′p76-508: Construct for The Expression of The GUS Gene Driven by the tms2Promoter Containing a ROS Operator (FIG. 6(B)).

The tms2 promoter is PCR amplified from genomic DNA of Agrobacteriumtumefaciens 33970 using the following primers:

sense primer: (SEQ ID NO:36) 5′-TGC GGA TGC ATA AGC TTG CTG ACA TTG CTAGAA AAG-3′ anti-sense primer: (SEQ ID NO:37) 5′-CGG GGA TCC TTT CAG GGCCAT TTC AG-3′The 352 bp PCR fragment is cloned into the EcoRV site of pBluescript,and sub-cloned into pGEM-7Zf(+). Two complementary oligos, ROS-OP1 (SEQID NO:34) and ROS-OP2 (SEQ ID NO:35), containing two ROS operators (initalics, below), are annealed together and cloned into pGEM-7Zf(+) as aBamHI/ClaI fragment at the 3′ end of the tms2 promoter. Thispromoter/operator fragment is then sub-cloned into pBI121 as aHindIII/XbaI fragment, replacing the CaMV 35S promoter fragment.

ROS-OP1: (SEQ ID NO:34) 5′-GAT CCT ATA TTT CAA TTT TAT TGT AAT ATA GCTATA TTT CAA TTT TAT TGT AAT ATA AT-3′ ROS-P2: (SEQ ID NO:35) 5′-CGA TTATAT TAC AAT AAA ATT GAA ATA TAG CTA TAT TAC AAT AAA ATT GAA ATA TA G-3′.

As a control, p76-507 comprising a tms2 promoter (without any operatorsequence) fused to GUS (FIG. 4(A)), is also prepared.

p74-501: Construct for The Expression of The GUS Gene Driven by TheActin2 Promoter Containing a ROS operator (FIG. 7B)).

The Actin2 promoter is PCR amplified from genomic DNA of Arabidopsisthaliana ecotype Columbia using the following primers:

Sense primer: (SEQ ID NO:38) 5′-AAG CTT ATG TAT GCA AGA GTC AGC-3′            SpeI Anti-sense primer: (SEQ ID NO:39) 5′-TTG ACT AGT ATCAGC CTC AGC CAT-3′The PCR fragment is cloned into pGEM-T-Easy. Two complementary oligos,ROS-OP1 (SEQ ID NO:34) and ROS-OP2 (SEQ ID NO:35), with built-in BamHIand ClaI sites, and containing two ROS operators, are annealed togetherand inserted into the Actin2 promoter at the BglII/ClaI sites replacingthe BglII/ClaI fragment. This modified promoter is inserted intopBI121vector as a HindIII/BamHI fragment.

As a control, p75-101, comprising an actin2 promoter (without anyoperator sequence) fused to GUS (FIG. 7(A)), is also prepared.

The various constructs are introduced into Arabidopsis, as describedabove, and transgenic plants are generated. Transformed plants areverified using PCR or Southern analysis. FIG. 8(A) show Southernanalysis of transgenic plants comprising a first genetic construct, forexample, p74-309 (35S-ROS operator sequence-GUS, FIG. 5(C))

Example 3 Crossing of Transgenic Lines Containing Fusion Constructs withTransgenic Lines Containing GUS Reporter Constructs

Transgenic Arabidopsis lines containing fusion constructs (secondgenetic constructs) are crossed with lines containing appropriatereporter (GUS) constructs (first genetic constructs). To perform thecrossing, open flowers are removed from plants of the reporter lines.Fully formed buds of plants of the repressor lines are gently opened andemasculated by removing all stamens. The stigmas are then pollinatedwith pollen from plants of the repressor lines and pollinated buds aretagged and bagged. Once siliques formed, the bags are removed, andmature seeds are collected. Plants generated from these seeds are thenused to determine the level of reporter gene (GUS) repression by GUSstaining. Levels of GUS expression in the hybrid lines are compared tothose of the original reporter lines. Plants showing a modified GUSexpression levels are further characterized using PCR, Southern andNorthern analysis.

Example 4 Preparation of a Chromatin Remodelling Factor

HDAC was used as an example of a chromatin remodelling factor that maybe isolated from an organism. Transcription factors that recruit histonedeacetylase (HDAC) to target promoters in Brassica napus were identifiedin vivo by screening a yeast two-hybrid library using the Arabidopsisthaliana HDA19 as bait. A cDNA clone that encodes a novel protein,bnKCP1, containing a kinase-inducible domain (KID) was identified.Southern blot analysis indicated that the bnKCP1 gene belongs to a smallgene family of at, least three members, and northern blot analysisshowed that it was strongly expressed in stems, flowers, roots andimmature siliques seeds, but not in leaf blades. In vitro proteinbinding assays showed that the protein is able to interact with bothHDA19 and histone acetyltransferase (HAT) and that the KID domain isrequired for this interaction with HDA19 and HAT in vitro. When assayedin vivo, bnKCP1 exerted modest activation of transcription of a reportergene in yeast.

The cAMP-responsive element (CRE) binding protein (CREB) binds to theCREB-binding protein (CBP) in response to extracellular stimuli thatinduce intracellular accumulation of secondary messengers Ca²⁺ and cAMP.The KID domain is highly conserved in the CREB family proteins, CREB,CREM and ATF-1 (Montminy, 1997). Each protein in this family has aserine phosphorylation site (RRPS ¹³³) within the KID domain, which isrecognised by protein kinase A (PK-A) that phosphorylates S¹³³. PK-A inturn is induced by outside stimuli that induce intracellularaccumulation of Ca²⁺ and cAMP. CREB binding activity is regulatedthrough S¹³³ phosphorylation, which leads to interaction of CREB withCBP. The KIX domain of CBP is required for interaction with the KIDdomain of CREB having a phosphorylated S¹³³ (see review Montminy, 1997).Interestingly, CBP possesses intrinsic HAT activity (Bannister andKouzarides, 1996; Ogryzko et al., 1996) suggesting that recruitment ofCBP to target promoters by the transcription activator CREB maycontribute to the transcriptional activation of CRE-dependent genes bythe involvement of histone acetylation at the genetic loci of targetgenes.

In Arabidopsis, a HAT gene encoding an ortholog of the yeast GCN5 wasfound to bind in vitro to two proteins similar to the yeast HAT-adaptorproteins ADA2, ADA2a and ADA2b (Stockinger et al., 2001). Moreover, thetranscription activator CBF1 was found to bind to both HAT and ADA2,indicating that these proteins might be recruited to targetcold-inducible genes by binding to CBF1 (Stockinger et al., 2001). Thefinding that the Arabidopsis ADA2 and GCN5 genes share similarity withtheir counterparts in yeast and humans suggests that chromatinremodelling complexes are conserved even among evolutionary distantorganisms.

Experimental Procedures

Brassica napus L. cv Cascade (winter type), Westar (spring type) andDES010 (spring type) were used for the isolation of genomic DNA andtotal RNA. Leaves, flowers, stems, siliques and immature seeds wereharvested from plants cultured in a controlled-environment greenhouseprogrammed for a photoperiod of 16 h day and 8 h night. Roots wereobtained by culturing sterilized seeds in 0.8% agar plates containing ½MS medium and 1% sucrose. For cold acclimation (4° C.), abscisic acid(250 μM), drought and high salt (850 mM NaCl) treatments, four-leafstage seedlings were treated and fourth fully expanded leaf blades wereharvested as described by Gao et al. (2002). LaCl3 and inomycintreatments were carried out by watering four-leaf stage plants with 20mM LaCl₃ and 10 μM inomycin, respectively. Plants were covered withSaran Wrap to slow evaporation.

Yeast Two-hybrid Screening and Cloning

A yeast two-hybrid cDNA library (Dr. Isobel Parkin, Agriculture andAgri-Food Canada Research Centre, Saskatoon) was constructed frompoly(A) mRNA isolated from the above-ground parts of the four-leaf stageseedlings of B. napus L. cv. DH12075 and cloned into a GAL4 AD(activation domain) vector pPC86 using the SuperScript Plasmid Systemfor cDNA Synthesis and Plasmid Cloning (GibcoL BRL).

To generate the pDBLeu-HDA19 construct, the entire coding region ofArabidopsis thaliana RPD3-type HDA19 cDNA (Accession # AF195547) was PCRamplified using PWO DNA polymerase (Roche) with a forward primer:

(SEQ ID NO: 44) 5′-GCGTCGACGATGGATACTGGCGGCAATTCGC-3′and a reverse primer:

(SEQ ID NO: 45) 5′-AGGCGGCCGCTTATGTTTTAGGAGGAAACGCC-3′.The identity of the PCR product was confirmed by DNA sequence analysisand inserted into the SalI and NotI sites of the Gal4 DB (DNA bindingdomain) vector pDBLeu in-frame with the GAL4 sequence and used as a baitto screen the B. napus cDNA library using PROQUEST Two-Hybrid System(GibcoL BRL).

Approximately 1×10⁶ transformants were subjected to the two-hybridselection on synthetic complete (SC) medium lacking leucine, tryptophanand histidine but containing 15 mM 3-amino-1,2,4-triazole (3AT®). Theexpression of the HIS3 reporter gene allowed colonies to grow on theselective medium and the putative His+ (3AT®) positive transformantswere tested for the induction of the two other reporter genes, URA3 andlacZ. The positive colonies were reassessed by retransformation assaysand the cloned cDNAs were identified by PCR and DNA sequence analysis.

Southern Blot Analysis

Total genomic DNA was isolated from the leaves of B. napus L. cv Westarusing a modified CTAB (cetyltriethylammonium bromide) extraction method(Gao et al., 2002). Briefly, 10 μg of total genomic DNA was digestedwith EcoRI, XbaI, HindIII, PstI, EcoRV and KpnI restrictionendonucleases, separated on a 0.8% agarose gel, transferred to Hybond-XLmembranes (Amersham Phamacia) and hybridized with the bnKCP1 openreading frame (ORF) labeled with [α-³²P]dCTP using random primerlabeling procedure. The DNA fragment to be used as a probe was isolatedfrom a 0.8% agarose gel and purified with a QIAquick Gel Extraction Kit(Qiagen), and the probe was purified with a ProbeQuant G-50 Micro Column(Amersham Phamacia). Hybridization was performed under high stringencyconditions (Gao, M.-J. et al., 2002).

Northern Blot Analysis

Total RNA was isolated from the tissues of B. napus L. cv DES010. Theseincluded leaves and stems of four-leaf stage seedlings, flowers,immature siliques of adult plants, and roots of cultured seedlings asdescribed by Gao et al. (2001). Probe labelling, hybridization, washingand membrane stripping were performed as described above in the Southernblot analysis Section.

Expression and Purification of Recombinant Gcn5 and HDA19

The full coding regions of the Arabidopsis HAT, Gcn5 (Dr. M. Thomashow,Michigan State University, MI), and HDA19 (Accession # AF195547) werePCR amplified, sequence analyzed and inserted in-frame with the GST(glutathione s-transferase) into the SalI and NotI sites of vectorpGEX-6P-2 (Amersham Pharmacia). The forward used for the amplificationof Gcn5 was:

(SEQ ID NO: 46) 5′-GCGTCGACGATGGACTCTCACTCTTCCCACC-3′and the reverse primer for Gcn5 was:

(SEQ ID NO: 47) 5′-GCGCGGCCGCCTATTGAGATTTAGCACCAGA-3′The forward primer for HDA19 was SEQ ID NO: 44, as listed above, and thereverse primer was:

(SEQ ID NO:48) 5′-GCGCGGCCGCTTATGTTTTAGGAGGAAACGC-3′.

Recombinant pGEX-6P-2 plasmids were used to transform E. coliBL21-CodonPlus (DE3)-RP competent cells (Stratagene). Expression andpurification under non-denaturing conditions were carried out asdescribed by Gao et al. (Gao, M.-J. et al., 2002). The GST-Gcn5 andGST-HDA19 fusion proteins were analyzed by 7.5% SDS-PAGE(SDS-polyacrylaride gel electrophoresis) and western blotting withrabbit anti-GST-Pi polyclonal antibody (Chemicon) using ECL Westernblotting analysis system (Amersham Pharmacia).

Generation of Deletion Mutants of bnKCP1

The two fragments, bnKCP1¹⁻¹⁶⁰ and bnKCP1¹⁻⁸⁰, and the entire codingregion of bnKCP1 DNA encoding amino acids 1-80, 1-160 and 1-215,respectively, were amplified by PCR and cloned into the HindIII and XhoIsites of pET-28-b vector (Novagen, Madison, Wis.). The primers used forthe amplification were as follows:

bnKCP1¹⁻¹⁶⁰ (240 bp): forward primer: (SEQ ID NO:49)5′-GCAAGCTTATGGCAGGAGGAGGACCAACT-3′, reverse primer: (SEQ ID NO:50)5′-CGCTCGAGCTCCTCCTCATCATTGTCTTC-3′; bnKCP1^(1–180) (480 bp): forwardprimer: (SEQ ID NO:49) 5′-GCAAGCTTATGGCAGGAGGAGGACCAACT-3′, reverseprimer (SEQ ID NO:51) 5′-CGCTCGAGATGAACAGGCAAAAGAGGCAT-3′; bnKCP1 (645bp): forward primer: (SEQ ID NO:49) 5′-GCAAGCTTATGGCAGGAGGAGGACCAACT-3′,reverse primer (SEQ ID NO:52) 5′-CGCTCGAGCTCaTCTTCTTCTTCTTCTTC-3′.In Vitro Protein Interaction Assays

Full-length bnKCP1 and truncated mutant bnKCP1¹⁻¹⁶⁰ and bnKCP1¹⁻⁸⁰proteins labeled with [³⁵S]methionine were produced using TNT-QuickCoupled Transcription/Translation System (Promega) according to themanufacture's instructions, with some modifications. A total of 1 μl ofRNase inhibitor (GibcoL BRL) and 1 μl of protease inhibitors set (Roche)were added to the lysate reaction. After incubation for 90 min at 30°C., RNase A was added to the reaction to a final concentration of 0.2mg/ml and incubated for 5 min at the same temperature.

In vitro protein interaction was detected with GST pulldown affinityassays as described by Ahmad et al. (1999) with some modifications.Briefly, 6 μg of GST or 4 μg of GST-fusion protein was incubated with 5μl of [³⁵S]Met-labeled translation mixture in 200 μl of bead-bindingbuffer (50 mM K-phosphate, pH 7.6,450 mM KCl, 10 mM MgCl₂, 10% glycerol,1% Triton X-100, 1% BSA and 1 μl of diluted 1:12 protease inhibitorsset) for 1 h at room temperature. After incubation, 20 μl of 50% slurryof glutathione-Sepharose beads containing 10 mg/ml of BSA and 4 μg ofEtBr was mixed with the reaction mixture followed by gentle rotation for1 h at 4° C. After washing six times with 1.2 ml of bead-binding bufferwithout BSA and EtBr but containing 12 μl of protease inhibitors set(Roche), the bound proteins were eluted with 30 μl of 2×SDS loadingbuffer, boiled for 2 min and analyzed by 12% SDS-PAGE. Afterelectrophoresis, the gels were dried, treated with Amplify (AmershamPharmacia) and subjected to fluorography.

In Vivo Protein Assays

The entire region of bnKCP1 and the two fragments, bnKCP1¹⁻¹⁶⁰ andbnKCP1¹⁻⁸⁰, were PCR amplified and cloned into the Sall and NotI sitesof pPC86 vector (GibcoL BRL) in-frame with the GAL4 AD sequences togenerate constructs pPC86-bnKCP1, pPC86-bnKCP1¹⁻¹⁶⁰ andpPC86-bnKCP1¹⁻⁸⁰. The oligonucleotide primers used in PCR amplificationwere as follows:

bnKCP1, bnKCP1¹⁻¹⁶⁰ and bnKCP1¹⁻⁸⁰ forward primer (SEQ ID NO:53)5′-GCGTCGACGATGGCAGGAGGAGGACCAACT-3′ bnKCP1 reverse primer (SEQ IDNO:54) 5′-GCGCGGCCGCCTCATCTTCTTCTTCTTCCTC-3′ bnKCP1¹⁻¹⁶⁰ reverse primer(SEQ ID NO:55) 5′-GCGCGGCCGCATGAACAGGCAAAAGAGGCAT-3′ bnKCP1¹⁻⁸⁰ reverseprimer (SEQ ID NO:56) 5′-GCGCGGCCGCCTCCTCCTCATCATTGTCTTC-3′For in vivo protein interaction assays, the MaV203 yeast cells carryingthe reporter gene lacZ and the construct pDBLeu-HDA19, in which theHDA19 was fused in-frame with GAL4DB, were transfected with either ofthe plasmids pPC86-bnKCP1, pPC86-bnKCP1¹⁻¹⁶⁰ and pPC86-bnKCP1¹⁻⁸⁰ or thevector alone. The expression of lacZ reporter gene was quantified bymeasuring the β-galactosidase activity using chlorophenolred-β-D-galactopyranoside (CPRG) according to the manufacturer'sinstructions (GibcoL BRL). Two yeast control strains A and B (GibcoLBRL) were used as negative and positive controls, respectively.Site-Directed Mutagenesis (SDM)

The QuickChange site-directed mutagenesis kit (Stratagene) was used toreplace the serine residue in the PK-A phosphorylation site (RRPS¹⁸⁸)within the KID domain with a glycine residue to generate bnKCP1G¹⁸⁸according to the manufacturer's instructions. The two oligonucleotideprimers used in SDM were as follows:

(SEQ ID NO:57) forward primer:5′-GATGTTCTTGCGAGGAGACCAGGATTCAAGAACAGAGCATTGAAG- 3′ (SEQ ID NO:58)reverse primer: 5′-CTTCAATGCTCTGTTCTTGAATCCTGGTCTCCTCGCAAGAACATC- 3′The introduced mutation was confirmed by DNA sequencing, and the mutatedbnKCP1G¹⁸⁸ was cloned into the HindIII and XhoI sites of pET-28b vectorto generate pET-bnKCP1G¹⁸⁸, which was then used for in vitro proteininteraction assays as described above.Transactivation Assay Using Yeast One-Hybrid System

Effector plasmids pDBLeu-bnKCP1¹⁻¹⁶⁰, pDBLeu-bnKCP1¹⁻⁸⁰,pDBLeu-bnKCP1⁸¹⁻²¹⁵ and pDBLeu-bnKCP1 were constructed by ligating thePCR-amplified fragments ΔbnKCP1¹⁻¹⁶⁰, ΔbnKCP1¹⁻⁸⁰, ΔbnKCP1⁸¹⁻²¹⁵ and thecoding region of bnKCP1 into the SalI/NotI sites of pDBLeu vector(GibcoL BRL) in-frame with the GAL4 DB sequence. The oligonucleotideprimers for PCR amplification were as follows:

bnKCP1 forward primer (SEQ ID NO:53)5′-GCGTCGACGATGGCAGGAGGAGGACCAACT-3′, bnKCP1 reverse primer (SEQ IDNO:54) 5′-GCGCGGCCGCCTCATCTTCTTCTTCTTCCTC-3′ bnKCP1¹⁻¹⁶⁰ forward primer(SEQ ID NO:53) 5′-GCGTCGACGATGGCAGGAGGAGGACCAACT-3′, bnKCP1¹⁻¹⁶⁰ reverseprimer (SEQ ID NO:55) 5′-GCGCGGCCGCATGAACAGGCAAAAGAGGCAT-3′ bnKCP1¹⁻⁸⁰forward primer (SEQ ID NO:53) 5′-GCGTCGACGATGGCAGGAGGAGGACCAACT-3′,bnKCP1¹⁻⁸⁰ reverse primer (SEQ ID NO:56)5′-GCGCGGCCGCCTCCTCCTCATCATTGTCTTC-3′ bnKCP1⁸¹⁻²¹⁵ forward primer (SEQID NO:59) 5′-GCGTCGACGCTAGGGTTGGCTTCATTGAGA-3′ bnKCP1⁸¹⁻²¹⁵ reverseprimer (SEQ ID NO:54) 5′-GCGCGGCCGCCTCATCTTCTTCTTCTTCCTC-3′The three reporter genes, lacZ, HIS3 and URA3, which were chromosomallyintegrated in the genome of MaV203 yeast cells were driven by unrelatedpromoters containing GAL4 DNA binding sites (GibcoL BRL). For transientassays, the effector constructs or the negative control vector pDBLeuwere transferred to the MaV203 yeast cells. The β-galactosidase activitywas measured using CPRG (chlorophenol red-β-D-galactopyranoside)according to the manufacturer's instructions (GibcoL BRL). The MaV203cells containing plasmids pDBLeu-HDA19 and pPC86-bnKCP1 were used as thepositive control. In addition, we used three yeast control strains A, B,and C (GibcoL BRL), which were developed to contain plasmid pairsexpressing fusion proteins with none, weak and moderately strongprotein-protein interaction strength, respectively.Transient Expression of the GUS-bnCKP1 Fusion Protein

The oligonucleotide primers for PCR amplification of the entire codingregion of bnKCP1 were as follows:

(SEQ ID NO:60) forward primer 5′-GCGAATTCATGGCAGGAGGAGGACCAACT-3′, (SEQID NO:61) reverse primer 5′-CGGAGCTCCTCaTCTTCTTCTTCTTCTTC-3′.The amplified sequence was cloned into the EcoRI and ScaI sites of thebinary vector p79-637, a derivative of the vector CB301, to generateconstruct p77-132, which contains GUS-bnKCP1 fusion under control of theCaMV35S promoter. The onion epidermal layers were transformed withAgrobacterium culture prepared as described by Kapila et al (1997) witha few modifications. Briefly, the onion inner epidermal layers werepeeled, placed into a culture of Agrobacterium tumefaciens strain GV3101pMP90 containing either p79-637, for GUS expression only, or p77-132 andsubjected to continuous vacuum of −85 IPA for 20 min. After incubationat 22° C. under 16 h light condition for 7 days the tissues were placedinto GUS staining solution [100 mM potassium phosphate buffer (pH 7.4),1 mM EDTA, 0.5 mM K₃Fe(CN)₆, 0.5 mM K₄Fe(CN)₆, 0.1% Triton X-100, 1 mM5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-gluc)], vacuum infiltratedfor 20 min at −85 kPa and incubated overnight at 37° C. To determine theintercellular location of nuclei, tissues were stained with thenucleus-specific 4′,6-diamidino-2-phenylindole (DAPI) solution (14 μg/mlDAPI, 0.1× PBS, 90% glycerol) (Varagona et al, 1991) and viewed under aZeiss microscope using both fluorescence and bright-field optics.Cloning of the B. napus KCP Protein

To identify proteins that interact with HDA19 in B. napus, the ORF ofArabidopsis HDA19 fused to the yeast Gal4 DNA binding domain was used asbait in a yeast two-hybrid screening of a B. napus cDNA library linkedto the yeast Gal4 activation domain. Several positive clones wereobtained on the basis of the induction of three yeast reporter genes,HIS3, URA3 and lacZ and DNA sequence analysis. One of these clones (963bp), pPC86-bnKCP1, encodes a 23.5 kDa protein that contains a putativekinase-inducible domain (KID)-like motif, and hence was designatedbnKCP1 (B. napus KID-containing protein 1).

Alignment of deduced amino acid sequences indicated that bnKCP1 sharesan 82% amino acid identity with atKCP, an Arabidopsis unknown 26.6 kDaprotein (AY088175, At5g24890). It also shares high similarity in theconserved region of approximately 55 amino acids (GKSKS domain) withother two other atKCP-like Arabidopsis unknown proteins, atKCL1(CAB45910, At4g31510) and atKCL2 (AAD23890, At2g24550) (FIG. 10A).

To estimate the bnKCP1 gene copy number in Brassica napus we carried outSouthern blot analysis on of total genomic DNA digested with restrictionendonucleases using the entire open reading frame of bnKCP1 for probingunder high stringency conditions (FIG. 12). Digestion with EcoRI (EI),HindIII (H), PstI (P), EcoRV (EV) and KpnI, none of which has a cuttingsite within bnKCP1, resulted in the detection of three bands, whereasdigestion with XbaI generated six bands, because of the existence of aninternal cutting site for XbaI in the bnKCP1 gene. This result indicatesthat bnKCP1 belongs to a small gene family of three members in theBrassica napus genomes.

Structural Features of the bnKCP1 Protein

The ORF of bnKCP1 gene codes for a 215 amino acid polypeptide product ofpolypeptide with several functional motifs (FIG. 11). Based on a searchof protein localization sites using PSORT program (seeURL:psort.nibb.acjp; Nakai and Kanehisa, 1992), bnKCP1 appears to be isa nuclear protein containing a pat7 nuclear localization signal (NLS)PLNKKRR (SEQ ID NO: 62; FIG. 10A, residues 127-133). Three acidic motifs(I, II and III) aid a serine-rich (S- rich) region (residues 34-58) mayfunction in transcription activation by bnKCP1 (Johnson et al., 1993).The charged motif GKSKS (residues 88-143), which is conserved in allfour protein orthologs (FIG. 10A), is rich in basic residues andencompasses the NLS. This suggests that this domain serve the mayfunction of a DNA-binding motif (FIG. 11). In addition, bnKCP1 isextremely hydrophilic (FIG. 11) suggesting bnKCP 1 is an active elementin the nuclear matrix.

Amino acid sequence analysis also revealed that bnKCP1 has a KID-likemotif (residues 161-215, FIG. 10A) with alpha structure at itsC-terminal region (FIG. 11). The KID is highly conserved in mammalianCREB protein family and functions in transactivation and protein binding(Montminy et al., 1997). The KID in bnKCP1 has a high similarity to theCREB family member ATF-1 (FIG. 10B, C) and contains a protein kinase A(PK-A) phosphorylation site (RRPS) that is conserved in the CREB familyof proteins (FIG. 10B).

Interaction of bnKCP1 with HDA19 and Gcn5

To confirm the interaction detected in the yeast two-hybrid systembetween the bnKCP1 protein and HDA19, GST pulldown assays were performedusing in vitro translated bnKCP1 labeled with [³⁵S]Methionine. ThebnKCP1 protein was tested for its ability to interact with recombinantGST-HDA19 or GST-GcnS fusions expressed in E. coli.

As shown in FIG. 13B, bnKCP1 bound to both GST-HDA19 and GST-Gcn5 fusionproteins, but not to GST alone. To reassess the interaction of bnKCP1with Gcn5 in vivo, the ORF of the Arabidopsis Gcn5 was fused to theyeast Gal4 DNA binding domain in pDBLeu vector and then used totransform yeast MaV203 cells expressing bnKCP1 fused to the yeast Gal4activation domain in pPC86 vector. The transformants showed induction ofthe three reporter genes, HIS3, URA3 and lacZ at a relatively lowerlevel when compared with the induction levels in transformants withbnKCP1 and HDA19 (data not shown). This result suggests that bnKCP1 hasa preference for binding to HDA19 in vivo.

To map the protein binding domain of the bnKCP1 protein, two C-terminaltruncated mutants of bnKCP1 lacking the KID domain were constructed.These are ΔbnKCP1¹⁻¹⁶⁰ (residues 1-160) and ΔbnKCP1¹⁻⁸⁰ (residues 1-80)as shown in FIG. 13A. These truncated mutants were assayed for, in vitrointeraction with the recombinant GST-HDA19 or GST-Gcn5 fusion proteins.The two mutant proteins, ΔbnKCP1¹⁻¹⁶⁰ and ΔbnKCP1¹⁻¹⁶⁰, exhibited nointeraction with either GST-HDA19 or GST-Gcn5 indicating that the KIDdomain of bnKCP1 protein is essential for binding to HDA19 and Gcn5.

The importance of the KID domain for protein binding was also determinedin vivo using the yeast two-hybrid system. MaV203 yeast cells wereco-transformed with pDBLeu-HDA19, and either pPC86-bnKCP1,pPC86-bnKCP1¹⁻¹⁶⁰, pPC86-bnKCP1¹⁻⁸⁰ or pPC86 alone (FIG. 13C ).β-galactosidase activity was reduced by at least 50% whenpDBLeu-expressing cells were transformed with plasmids expressing eitherΔbnKCP1¹⁻¹⁶⁰ or ΔbnKCP1¹⁻⁸⁰, both of which lacked KID, as compared tothe full-length bnKCP1. This finding demonstrates that KID is criticalfor bnKCP1 interaction with HDA19 in vivo.

To investigate the importance of S¹⁸⁸ for bnKCP1 interaction with HDA19,the S¹⁸⁸ residue in bnKCP1 was mutated to G¹⁸⁸ using site-directedmutagenesis to obtain bnKCPG¹⁸⁸ protein (FIG. 14). This mutated proteinwas then tested for binding to HDA19 in vitro. When compared to bnKCP1,the mutated protein, bnKCPG¹⁸⁸, has significantly reduced binding toHDA19 (FIG. 14). This confirms that S¹⁸⁸ is essential for optimalinteraction between bnKCP1 and HDA19.

Expression Pattern of the bnKCP1 Gene

The expression pattern of the bnKCP1 gene was analyzed by Northern blotanalysis of total RNA extracted from various organs of B. napus. Asshown in FIG. 15, two transcripts of similar sizes appear to hybridizeto bnKCP1, indicating the existence of two homologs of bnKCP1 mRNAs inB. napus. These transcripts accumulated at high levels in flowers,roots, stems and immature siliques, and at low levels in leaves withpetioles, but were undetectable in leaf blades (FIGS. 15, 16).

To investigate the pattern of bnKCP1 expression in response toenvironmental stress conditions, total RNA was isolated from leaf bladesof four-leaf stage B napus seedlings that were exposed to lowtemperature (4° C.), drought, high salt (NaCl), and ABA treatment, andused for northern blot analysis using a bnKCP1 probe. Transcripts ofboth bnKCP1 homologs accumulated in leaves in response to coldtreatment. The lower size (˜0.9 kb) transcript appears to be inducedwithin 4 h of cold treatment and about 4 h earlier than the highermolecular weight (1.1 kb) one (FIG. 16A). The bnKCP1 transcript appearsto accumulate in response to low temperature (4° C.), but expression wasnot detected in leaf blades of plants grown under drought condition forup to 4 days, high salt stress for up to 11 days, or upon exogenousapplication of ABA for up to 8 hours (data not shown). Expression ofbnKCP1 in the stems, was repressed upon cold treatment (FIG. 16A),suggesting the response of bnKCP1 transcript to low temperature or therecruitment of HDA19 and HAT to the promoters of cold responsive genesis organ specific.

Since cold acclimation is known to be associated with elevated levels ofintracellular concentrations of Ca²⁺, tests to determine whether Ca⁺²has any effect on bnKCP1 expression were performed. Northern blotanalysis was performed using total RNA isolated from leaves of seedlingstreated with Ca²⁺ channel blocker LaCl₃ and the Ca²⁺ ionophore inomycinat room temperature. Induction of bnKCP1 expression upon treatment withinomycin was rapid (2 hrs) but short-lived. The bnKCP1 transcript wasundetectable in leaves of seedlings treated with the LaCl₃ (FIG. 16B).

Transcription Activation by bnKCP1

To determine whether bnKCP1 functions as a transcription activator,transactivation experiments were carried out in yeast. A yeast straincarrying three reporter genes, lacZ, HIS3 and URA3, driven by promotersfused to GAL4 DNA binding sites and independently integrated into theyeast genome were transfected with the effector plasmid pDBLeu-bnKCP1comprising bnKCP1 fused to the GAL4 DB under the control of ADHpromoter. The effector stimulated β-galactosidase activity about 8-foldrelative to either GAL4 DB alone or yeast control strain A that containsplasmid pairs expressing fusion proteins without protein-proteininteraction. A similar result was obtained when the yeast cells wereco-transformed with the positive control plasmids pDBLeu-HDA19 andpPC86-bnKCP1 identified by the two-hybrid selection (FIG. 17A). Reportergenes HIS3 and URA3 were also modestly activated by bnKCP1 (data notshown). Based on these findings, it can be concluded that bnKCP1 exertstransactivation of target genes in Brassica napus.

These data demonstrate the isolation of a plant protein that contains aputative KID domain, which interacts with both GCN5 (HAT) and HDA19.bnKCP1 was highly expressed in all organs tested, except leaf blades,where it was induced in response to cold acclimation, which alsoresulted in repressing its expression in stems. Furthermore, bnKCP1exerts transcription activation of a reporter gene when tested in yeast,indicating the function of bnKCP1 as a transcription factor.

To map the transactivation domain of the bnKCPP1 protein, one N-terminaltruncated mutant of bnKCP1, ΔbnKCP1⁸¹⁻²¹⁵, and two C-terminal truncatedmutants, ΔbnKCP1¹⁻¹⁶⁰ and ΔbnKCP1¹⁻⁸⁰ (FIG. 17B) were generated and usedin in vivo transactivation assays in yeast. As shown in FIG. 17C,deletion of the KID or GKSKS domains had no significant influence onβ-galactosidase activity, whereas deletion of the N-terminus resulted inapproximately 65% reduction in β-galactosidase activity.

Nuclear Localization of the bnKCP1 Protein

Structural and functional analyses showed bnKCP1 to have featurestypical of transcription factors. To confirm that bnKCP1 is a nuclearproteins, onion epidermal cell layers were transformed with constructsfor the expression of either a GUS-bnKCP1 fusion or GUS alone (FIG. 18).Using an Agrobacterium-mediated transformation method (Kapila et al,1997). As shown in FIG. 18, GUS activity was visualized exclusively inthe cytoplasm of control onion cell layers. In contrast, a blueprecipitate was localized in the nuclei of cell layers transformed withGUS-bnKCP1 fusion construct, although there was still a certain amountof cytoplasm staining, indicating that at least some targeting to thenucleus occurs with the fusion protein.

Expression of bnKCP1 is Organ-Specific

The bnKCP1 gene appears to be part of a multigene family of threemembers based on Southern blot hybridization. Northern blot analysesshowed that two members of this gene family are of similar transcriptsizes and expression patterns. This is consistent with information aboutbnKCP1 orthologs in Arabidopsis, where there are one atKCP (At5g24890)and two atKCP-like members (At4g31510 and At2g24550) of similar sizesranging from 1 kb to 1.2 kb. Northern blot analysis revealed that bnKCP1mRNA was expressed in flowers, roots, stems and immature siliques (FIG.14). The transcript accumulation, however, was undetectable in leafblades of B. napus seedlings, suggesting tissue/organ-specificexpression of the bnKCP1 gene. However, cold treatment induced bnKCP1expression in leaves, but repressed it in stems.

The KID Domain is Conserved in bnKCP1

Structural analysis of the bnKCP1 protein revealed that it was astrongly hydrophilic protein (23.5 kDa, pI 4.2) and had characteristicfeatures of a transcription factor, including a putative nuclearlocalization signal (NLS), a putative basic DNA binding domain, putativeacidic activation domains and a protein-protein interaction domain.

An important structural feature of bnKCP1 is the presence of a putativekinase-inducible domain (KID) with alpha secondary structure at theC-terminal region. The KID domain was first identified in mammalian CREBfamily members CREB, CREM and ATF-1. The KID domain in mammalian CREB isinvolved in at least two functions, interaction with CBP/p300 and thesite for protein kinase A (PK-A) phosphorylation of S¹³³ (Montminy etal., 1997; Gonzalez et al., 1991; Quinn, 1993; Chrivia et al., 1993;Shaywitz et al., 2000). Similar to its counterpart in CREB, which isinvolved in protein binding, the KID domain of bnKCP1 is required forbinding to both HDA19 and GCN5 in vitro and in vivo. The ability ofbnKCP1 to interact with HDA19 indicates that bnKCP1-mediatedtranscription control requires direct or indirect recruitment of thesetranscription regulators to promoter regions of target genes regulatedby bnKCP1.

Phosphorylation of CREB at Ser¹³³ is required for the interaction ofCREB via its KID with CBP and for CREB to activate transcription inresponse to some extracellular stimuli (Gonzalez et al, 1989; Chrivia etal., 1993). The KID domain in bnKCP1 also contains a putative PK-Aphosphorylation site (RRPS¹⁸⁸), which corresponds to the RRPS¹³³ inmammalian CREB.

Intracellular Level of Ca⁺² Affect bnKCP1 Expression

In mammalian cells, outside stimuli that increase intracellularconcentrations of Ca²⁺ or cAMP induce the expression of not only PK-A,but also the CREB gene (Meyer et al., 1993). Therefore, tests todetermine whether conditions that increase intracellular concentrationsof Ca²⁺ would induce bnKCP1 expression were done. B. napus seedlingswere subjected to one of two treatments, cold or inomycin. Coldacclimation is known to increase intracellular Ca²⁺ concentrations(Monroy and Dhindsa, 1995; Knight et al., 1996), and inomycin is a knowncalcium ionophore that increases Ca²⁺ influx (Hurley et al., 1996).These treatments resulted in the induction of bnKCP1 expression tovarying degrees (FIG. 16), which indicated that bnKCP1 is induced byhigh intracellular Ca⁺² concentrations.

These results suggest a molecular mechanism by which bnKCP1 functions asa transcription factor to regulate gene expression by recruiting HDAC tothe promoter regions of target genes.

Example 5 Characterization of the Recruitment Factor SCL1 and itsInteraction with the Chromatin Remodelling Factor HDA19

To search for transcription factors additional that recruit histonedeacetylase (HDAC) to target promoters in Brassica napus, a yeasttwo-hybrid library was screened using the Arabidopsis thaliana HDA19 asbait. This screening resulted in the isolation of a cDNA clone thatencodes a SCARECROW-like protein, BnSCL1, which contains a number ofputative functional motifs typical of the GRAS family of transcriptionfactors. Southern blot analysis indicated that the BnSCL1 gene belongsto a small gene family of about three members. In vitro and in vivoprotein interaction assays revealed that BnSCL1 interacts physicallywith HDA19 through the VHIID domain. BnSCL1 also exerted strongtransactivation of the lacZ reporter gene in yeast, and both N- andC-terminal regions are critical for the transient expression.Quantitative RT-PCR and RNA gel blot analysis showed that BnSCL1 wasexpressed at relatively high level in roots, moderate level in flowers,weak in mature leaves and stems, and barely detectable in immaturesiliques. The accumulation of BnSCL1 transcript was regulated by 2,4-Din shoots, roots and matured leaves. Furthermore, the response of BnSCL1to 2,4-D was modulated by histone deacetylase HDA19. These resultsstrongly suggest a molecular mechanism by which BnSCL1 functions as atranscription factor to regulate gene expression by recruiting HDAC tothe promoter regions of auxin-responsive genes.

Plant Materials

Brassica napus L. cv. DH12075 was used for DNA and total RNA isolation.Leaves, flowers, stems, siliques and immature seeds were harvested fromplants cultured in a controlled-environment greenhouse programmed for aphotoperiod of 16 h day and 8 h night. Roots were obtained by culturingsterilized seeds in 0.8% agar plates containing ½ MS medium (Murashigeand Skoog, 1962) and 1% sucrose.

Tissue Treatment

In exogenous applied auxin treatments, four-leaf stage seedlings grownat 20° C. were treated with a foliar spray containing 1 mM 2,4-D and 50mM sodium phosphate, pH 7.5. The four leaves were collected at 30 min,60 min and 180 min after the first foliar application of 2,4-D. For themeasurement of response of shoots and roots to auxin, sterilized seedswere germinated on plates in a growth chamber with continuous light at20° C., and 10 dpg seedlings were supplied with varied concentration of2,4-D. In the auxin transport inhibition experiments, 9 dpg seedlingswere incubated in the medium supplemented with 50 μM NPA dissolved in0.1% DMSO for 24 h before the 2,4-D treatment. For the HDAC inhibitortreatments, 10 mM sodium butyrate was added onto the growth medium andincubated for 24 h followed by exogenous 2,4-D application at variedconcentrations.

Yeast Two-Hybrid Screening and Cloning

A yeast two-hybrid cDNA library was constructed from seedlings of B.napus L. cv. DH12075 and screened using a Arabidopsis thaliana RPD3-typeHDAC (HDA19) as bait, with the methods of PROQUEST Two-Hybrid System(GibcoL-BRL) as previously described by Gao et al. (2003). The positivecolonies were reassessed with retransformation experiments and confirmedwith in vitro protein interaction assays, and the cloned cDNAs wereidentified by PCR and DNA sequence analysis.

Gel Blot Analysis

Total genomic DNA was extracted from the leaves of four-leaf stage B.napus using a modified CTAB (cetyltriethylammonium bromide) extractionmethod, and DNA gel blots were prepared and hybridized with the BnSCL1open reading frame labeled with [α-³²P]dCTP using random primer labelingprocedure as described by Gao et al. (2003). Total RNA was isolatedusing hot phenol method with the first extraction for 30 sec at 80° C.as previously described (Gao et al. 2002). RNA was isolated from varioustissues, including leaves and stems of four-leaf stage seedlings,flowers, immature seeds and siliques of adult plants, and roots ofcultured seedlings.

Quantitative RT-PCR

Total RNA extracted as described above was treated with AmplificationGrade Deoxyribonuclease I (GibcoL BRL) following the manufacture'sinstructions. The RNA samples were then directly used for reversetranscription prior to amplification without purification. The RT-PCRwas quantitatively performed and completed in a one-step reaction usingSuperscript One-Step RT-PCR System (GibcoL BRL) as described by Gao etal. (2002). Gene-specific sense and anti-sense primers used to generatea 960 bp fragment of Brassica napus Actin, as an internal standard, wereas described in Gao et al., (2002). Gene-specific primers for thegeneration of BnSCL1, BnIAA1 and BnIAA12 fragments were as follows:

BnSCL1 (435 bp) (SEQ ID NO:84) sense: 5′-GATGGACGAACATGCCATGCGTTCCA-3′(SEQ ID NO:85) anti-sense: 5′-CGCTCGGATCTTCTGAACAAT-3′ BnIAA1 (537 bp)(SEQ ID NO:86) sense: 5′-CCACGCGTCCGGTACGATGAT-3′ (SEQ ID NO:87)anti-sense: 5′-GAAGTTGAGAAATGGTTTATGA-3′ BnIAA12 (659 bp) (SEQ ID NO:88)sense: 5′-ACGCTGGTGCTTCTCCTCCTC-3′ (SEQ ID NO:89) anti-sense:5′-AAAACCCATTAGAAGAACCAAGAA-3′

BnIAA1 and BnIAA12 are clones ML2798 and ML4744, which are homologs ofArabidopsis IAA1 and IAA12, respectively, and were identified in adatabase of Brassica napus ESTs that were generated at the SaskatoonResearch Centre of Agriculture and Agri-Food Canada (seeURL:.brassica.ca).

Expression and Purification of Recombinant HDA19

The open reading frame (ORF) of the HDA19 was PCR amplified, sequenceanalyzed, inserted in-frame with the GST (glutathione s-transferase)into the vector pGEX-6P-2 (Amersham Pharmacia), and transformed into E.coli BL21-CodonPlus (DE3)-RP competent cells (Stratagene) as previouslydescribed (Gao et al., 2003). The recombinant HDA19 protein wasexpressed and purified under non-denaturing conditions as described byGao et al, 2002). The GST-HDA19 fusion protein was analyzed by westernblotting with rabbit anti-GST-Pi polyclonal antibody (Chemicon) usingECL Western blotting analysis system (Amersham Pharmacia).

In Vitro Protein Interaction Assays

The entire coding region of BnSCL1 and four fragments, BnSCL1¹⁻³⁵⁸,BnSCL1¹⁻²⁶¹, BnSCL1¹⁻²¹⁷, and BnSCL1¹⁻¹⁴⁵ encoding amino acids 1-434,1-358, 1-261 and 1-217, respectively, were amplified by PCR and clonedinto the HindIII and XhoI sites of the expression vector pET-28b(Novagen) in-frame with the His-Tag sequence. The primers used foramplification were as follows:

Forward primer for BnSCL1, BnSCL1¹⁻³⁵⁸, BnSCL1¹⁻²⁶¹, BnSCL1¹⁻²¹⁷ andBnSCL1¹⁻¹⁴⁵: (SEQ ID NO:90) 5′-GCAAGCTTATGGACGAACATGCCATGCGTTCCA-3′Reverse primer for BnSCL1: (SEQ ID NO:91)5′-CGCTCGAGAAAGCGCCACGCTGACGTGGC-3′ Reverse primer for BnSCL1¹⁻³⁵⁸: (SEQID NO:92) 5′-CGCTCGAGCGCGGAGATCTtCGGACGTAA-3′ Reverse primer forBnSCL1¹⁻²⁶¹: (SEQ ID NO:93) 5′-CGCTCGAGCCTAATCGCCTTGAAAGATAA-3′ Reverseprimer for BnSCL1¹⁻²¹⁷: (SEQ ID NO:94)5′-CGCTCGAGCGCCACAACCGCCGTGACTCT-3′ Reverse primer for BnSCL1¹⁻¹⁴⁵: (SEQID NO:95) 5′-CGCTCGAGCGCTCGGATCTTCTGAACAAT-3′.

The TNT-Quick Coupled Transcription/Translation System (Promega) wasused to produce the full-length BnSCL1 protein and the truncated mutantsΔBnSCL1¹⁻³⁵⁸, ΔBnSCL1¹⁻²⁶¹, ΔBnSCL1¹⁻²¹⁷ and ΔBnSCL1¹⁻¹⁴⁵ labeled with[³⁵S]methionine as previously described (Gao et al., 2003). In vitroprotein interaction was detected with GST pulldown affinity assays asdescribed by Ahmad et al. (1999) and Gao et al., (2003).

In Vivo Protein Interaction Assays

The six DNA fragments, BnSCL1¹⁻³⁵⁸, BnSCL1¹⁻²⁶¹, BnSCL1¹⁻²¹⁷,BnSCL1¹⁻¹⁴⁵, BnSCL1¹⁴⁶⁻³⁵⁸ and BnSCL1²¹⁸⁻⁴³⁸ and the ORF of BnSCL1encoding amino acids 1-358, 1-261, 1-217, 1-415, 146-358, 218-434 and1434, respectively, were PCR amplified and cloned into the SalI and NotIsites of pPC86 vector (GibcoL BRL) in-frame with the GAL4 AD sequencesto generate constructs pPC86-BnSCL1¹⁻³⁵⁸, pPC86-BnSCL1¹⁻²⁶¹,pPC86-BnSCL1¹⁻²¹⁷, pPC86-BnSCL1¹⁻¹⁴⁵, pPC86-BnSCL1¹⁴⁶⁻³⁵⁸,pPC86-BnSCL1²¹⁸⁻⁴³⁸ and pPC86-BnSCL1. PCR amplification was carried outusing the following primers:

Forward primer for BnSCL1, BnSCL1¹⁻³⁵⁸, BnSCL1¹⁻²⁶¹, BnSCL1¹⁻²¹⁷ andBnSCL1¹⁻¹⁴⁵: (SEQ ID NO:96) 5′-GCGTCGACGATGGACGAACATGCCATGCGTTCCA-3′Forward primer for BnSCL1¹⁴⁶⁻³⁵⁸: (SEQ ID NO:97)5′-GCGTCGACGATTAAGGAGTTTTCCGGTATA-3′ Forward primer for BnSCL1²¹⁸⁻⁴³⁴:(SEQ ID NO:98) 5′-GCGTCGACGGAGGATTGCGCCGTCGAGACG-3′ Reverse primer forBnSCL1 and BnSCL1²¹⁸⁻⁴³⁴: (SEQ ID NO:99)5′-GCGCGGCCGCAAAGCGCCAGGCTGACGTGGC-3′ Reverse primer for BnSCL1¹⁻³⁵⁸:(SEQ ID NO:100) 5′-GCGCGGCCGCCGCGGAGATCTTCGGAC GTAA-3′ Reverse primerfor BnSCL1¹⁻²⁶¹: (SEQ ID NO:101) 5′-GCGCGGCCGCCCTAATCGCCTTGAAAGATAA-3′Reverse primer for BnSCL1¹⁻²¹⁷: (SEQ ID NO:102)5′-GCGCGGCCGCCGCCACAACCGCCGTGACTCT-3′ Reverse primer for BnSCL1¹⁻¹⁴⁵:(SEQ ID NO:103) 5′-GCGCGGCCGCCGCTCGGATCTTCTGAACAAT-3′ Reverse primer forBnSCL1¹⁴⁶⁻³⁵⁸: (SEQ ID NO:100) 5′-GCGCGGCCGCCGCGGAGATCTTCGGACGTAA-3′.

For in vivo protein interaction assays, the MaV203 yeast competent cellscarrying the lacZ reporter gene were co-transfected with the constructpDBLeu-HDA19, in which the HDA19 was fused in-fame with GAL4 DB andeither of the plasmids pPC86-BnSCL1, pPC86-BnSCL1¹⁻³⁵⁸,pPC86-BnSCL1¹⁻²⁶¹, pPC86-BnSCL1¹⁻²¹⁷, pPC86-BnSCL1¹⁻¹⁴⁵,pPC86-BnSCL1¹⁴⁶⁻³⁵⁸, pPC86-BnSCL1²¹⁸⁻⁴³⁸ and or the vector pPC86 alone.The expression of lacZ reporter gene was quantified by measuring theβ-galactosidase activity using CPRG (chlorophenolred-β-D-galactopyranoside) according to the manufacturer's instructions(GibcoL BRL). Three yeast control strains A, B, and C (GibcoL BRL) thatcontain plasmid pairs expressing fusion proteins with none, weak andmoderately strong interaction strengths, respectively, were used ascontrols.

Transactivation Assay

MaV203 yeast cells expressing the lacZ reporter gene driven by apromoter containing GAL4 DNA binding sites (GibcoL BRL) were transformedwith the pDBLeu-bnKCP1¹⁻¹⁶⁰, pDBLeu-bnKCP1¹⁻⁸⁰, pDBLeu-bnKCP1⁸¹⁻²¹⁵ andpDBLeu-bnKCP1. These vectors were constructed by ligating thePCR-amplified fragments, ΔBnSCL1¹⁻³⁵⁸, ΔBnSCL1¹⁻²⁶¹, ΔBnSCL1¹⁻²¹⁷,ΔBnSCL1¹⁻¹⁴⁵, ΔBnSCL1¹⁴⁶⁻³⁵⁸ and ΔBnSCL1²¹⁸⁻⁴³⁴ and the entire codingregion of BnSCL1, respectively, into the SalI and NotI sites of thevector pDBLeu (GibcoL BRL) in-frame with the GAL4 DB sequence. Theoligonucleotide primers for the amplification were the same as thoseused for the in vivo protein interaction assays. The β-galactosidaseactivity was measured using CPRG according to the manufacturer'sinstructions (GibcoL BRL). In addition to the yeast strains A, B and C,the yeast strains D (GibcoL BRL) that contain plasmid pairs expressingfusion protein with strong interaction strength was used as controls.

Cloning and Sequence Analysis of the BnSCL1 Gene

HDAC or HAT is recruited to specific loci by large protein complexesmade up of transcription activators/co-activators andrepressors/co-repressors, respectively (See reviews Kuo and Allis, 1998;Meyer, 2001). Identification of these transcription regulatory proteinsthat interact with HDAC or HAT is a direct approach to defining nuclearfactors that recruit these chromatin remodelling regulators to theirtarget promoters and hence affect the expression of the target genes. Toisolate proteins that bind to HDAC in B. napus, the ORF of Arabidopsisthaliana HDA19 fused to the yeast Gal4 DNA binding domain was used asbait in a yeast two-hybrid screening of a B. napus cDNA library linkedto the yeast Gal4 activation domain. A number of positive clones wereobtained on the basis of the induction of three yeast reporter genesHIS3, URA3 and lacZ followed by retransformation and sequencinganalysis. One of these clones encodes a 51.2 kDa protein with pI 5.1,designated BnSCL1 (Brassica napus SCARECROW-like protein 1; SEQ IDNO:81). As shown in FIG. 20, BnSCL1 contains several domains of theSCARECROW (SCR) family of transcription factors (Laurenzio et al.,1996).

Sequence analysis revealed that BnSCL1 cDNA (2781 bp) contains two openreading frames (ORFs). The first ORF (ORF1) encodes BnSCL1, apolypeptide of 461 amino acid residues starting at 82 bp from the 5′end, and ORF2 codes for a polypeptide of 281 amino acids starting at1687 bp from the 5′ end. The linking region of the two ORFs is a shortsequence of 200 bp. Database search using NCBI blast program (Altschulet al., 1997) indicated that the deduced amino acid sequence encoded byORF2 was similar to the human polyposis coli region hypothetical proteinDP1 (accession number A39658), which contains a TB2_DP1_HVA22 domain.However, the GENESCAN program (Burge and Karlin, 1997) predicts that the2781 bps of BnSCL1 cDNA encodes one polypeptide only, i.e. the deducedamino acid sequence of ORF1.

Comparison of the deduced BnSCL1 amino acid sequence to the NCBI (seeURL:.ncbi.nln.nih.gov) and TAIR (see URL:arabidopsis.org) databasesresults in a list of proteins with considerable similarity (FIG. 21).According to the NTI computer program (InforMax, Inc.), BnSCL1 shares an89% amino acid identity with AtSCL15 (Pysh et al., 1999) or VHS5(Silverstone et al., 1998), an Arabidopsis SCARECROW-like protein(accession number Z99708, At4g36710), while it is 37% identical to AtSCR(accession number U62797). Interestingly, it also shares high similarity(66% sequence identity) with a tomato (Lycopersicon esculentum) protein(accession number AF273333), a member of the GRAS/VHIID protein family,encoded by the Lateral suppressor gene (Ls) (Schumacher et al., 1999)(FIG. 20). Consistent with these data, phylogenetic analysis usingeither NTI Vector or DNA Star program classified BnSCL1, AtSCL15 andLsSCL (Ls) in the same subgroup (FIG. 21).

The BnSCL1 copy number in B. napus was estimated using DNA gel blotanalysis on total genomic DNA digested with restriction endonucleasesand hybridized with the ORF of BnSCL1 under high stringency conditions(FIG. 22). Digestion with EcoRI, XbaI, HindIII, PstI and KpnI resultedin the detection of about three bands, whereas digestion with EcoRVgenerated approximately six bands due to the existence of an internalcutting site for EcoRV within the BnSCL1 gene. This result indicatesthat BnSCL1 belongs to a small gene family of approximately threemembers in the B. napus genomes.

BnSCL1 is a Member of GRAS/VHIID Family

The BnSCL1 gene encodes a polypeptide of 461 amino acids with severalsuggestive functional domains or motifs (FIG. 20). It has two MATα2-like nuclear localization signals (NLSs) (residues 169-173 and436440) (Raikhel, 1992). It also has a LXXLL motif (¹⁴⁸LGSLL¹⁵² (SEQ IDNO:104)) that was shown to mediate interaction of transcriptioncoactivators with nuclear receptors (Heery et al., 1997). Amino acidsequence analysis also revealed that BnSCL1 has the characteristicstructure for GRAS/VHIID regulatory proteins (Pysh et al., 1999),including a VHIID motif that encompasses a putative NLS, two leucineheptad repeats (LHRs) that surround the conserved VHIID motif, a PFYREmotif and a C-terminal SAW motif that encompasses a putative NLS (FIG.20). The LHRI-VHIID-LHRII region has been thought to function inprotein-protein and DNA-protein interactions (Pysh et al., 1999).

BnSCL1 Interacts Physically with HDA19 in Vitro and in Vivo

To confirm the interaction of BnSCL1 protein with HDA19 that wasdetected in the yeast two-hybrid system, GST pulldown affinity assayswere carried out using in vitro-translated BnSCL1 labeled with[³⁵S]Methionine. The BnSCL1 protein was tested for its binding abilityto GST-HDA19 fusion protein that was expressed in Escherichia coli andpurified under non-denaturing conditions. As shown in FIG. 23, BnSCL1bound to recombinant HDA19 protein, while it did not bind to GST alone(data not shown).

To map the protein binding domain of the BnSCL1 protein, four C-terminaltruncated mutants of BnSCL1 lacking either of the SWA, PFYRE, LHRII orVHllD motif (FIG. 23 a) were constructed. These truncated mutants wereassayed for in vitro interaction with the recombinant HDA19 protein. Asshown in FIG. 23 b, the mutant proteins exhibited interaction withGST-HDA19 fusion protein with the truncation from C-terminal end untilthe VHIID region was deleted, indicating that the VHIID domain isessential for BnSCL1 protein binding to HDA19.

The requirement of the VHIID domain for protein-protein interaction wasalso demonstrated in vivo using the yeast two-hybrid system (FIG. 24).MaV203 yeast cells were co-transformed with plasmid pDBLeu-HDA19 andeither pPC86-BnSCL1, pPC86-BnSCL1¹⁻³⁵⁸, pPC86-BnSCL1¹⁻²⁶¹,pPC86-BnSCL1¹⁻²¹⁷, pPC86-BnSCL1¹⁻¹⁴⁵, pPC86-BnSCL1¹⁴⁶⁻³⁵⁸,pPC86-BnSCL1²¹⁸⁻⁴³⁸ and or the vector pPC86 alone. Althoughβ-galactosidase activity was reduced by at least 50% whenpDBLeu-expressing cells were transformed with plasmids expressing eitherof the six mutants of BnSCL1 protein, as compared to the wild typeBnSCL1, the transformants with plasmids expressing eitherpPC86-BnSCL1¹⁻¹⁴⁵ or pPC86-BnSCL1²¹⁸⁻⁴³⁸, both of which lacked VHIIDmotif, showed a further at least 50% reduction in β-galactosidaseactivity as compared to the other mutants. This finding indicates thatVHIID domain is critical for BnSCL1 interaction with HDA19 in vivo.

BnSCL1 Activates Transcription of a Reporter Gene in Yeast

To further characterize the biological function of BnSCL1, its functionsas a transcription activator was investigated. Transactivationexperiments were performed in yeast (FIG. 25), whereby a yeast straincarrying three reporter genes, lacZ, HIS3 and URA3, driven by promotersfused to GAL4 DNA binding sites and independently integrated into theyeast genome were transfected with the effector plasmid pDBLeu-BnSCL1comprising BnSCL1 fused to the GAL4 DB under the control of the ADHpromoter. Transformation with the effector plasmid resulted inincreasing β-galactosidase activity similar with yeast strain D thatcontains plasmid pairs expressing fusion proteins with strongprotein-protein interaction and approximately 20-fold relative to eithervector pDBLeu alone or yeast control strain A, which contains plasmidpairs expressing fusion proteins without protein-protein interaction(FIG. 25). Reporter genes HIS3 and URA3 were also stronglytransactivated by BnSCL1 protein (data not shown). These resultsindicate that BnSCL1 significantly exhibits transcription activatoractivity in yeast.

To map the transactivation domain of the BnSCL1 activator, a series ofdeletion mutants of BnSCL1 protein were generated (FIG. 25 a) and usedin vivo transactivation assays in yeast. As shown in FIG. 6 b, either ofthe deletions from C-terminal of BnSCL1 or any truncation from theN-treminal resulted in a decrease of at least 85% in β-galactosidaseactivity relative to the wild type BnSCL1 protein. This demonstratesthat the transactivation domain of bnKCP1 may reside in both the N- andC-terminal regions.

BnSCL1 Gene is Expressed Mainly in Roots

The expression pattern of the BnSCL1 gene was analyzed by RNA gel blotanalysis and quantitative RT-PCR using total RNA extracted from variousorgans of B. napus (FIG. 26). As shown in FIG. 26 a, there were twobnSCL1 transcripts of 1.6 kb and 2.8 kb in the RNA blot probed with theORF of BnSCL1, suggesting the existence of either two species of BnSCL1cDNA produced by alternative splicing in B. napus genome or a BnSCL1homologue cross-hybridizing to the probe. Both of them accumulated athighest levels in roots, whereas its expression was weak in flowers andstems, and undetectable in leaves and siliques. Results obtained usingquantitative RT-PCR analysis (FIG. 26 b) were consistent with thoseobtained with northern blotting. In addition, RT-PCR analysis revealedstrong expression in seedling shoots (FIG. 26 b). This expressionpattern is similar to that of Arabidopsis SCR gene (Laurenzio et al.,1996) and to those of most SCLs (Pysh et al., 1999). This suggests thatBnSCL1 and SCR may share similar functions in the regulation of rootdevelopment.

BnSCL1 Responds to Auxin Treatment

The plant hormone auxin plays an important role in cell division, cellelongation, cell differentiation, lateral root initiation andgravitropism (Davies, 1995; Berleth and Sachs, 2001; Liscum andStowe-Evans, 2000). Recent studies have demonstrated that auxindistribution organizes the pattern and polarity in the root meristem(Sabatini et al., 1999). To determine whether the dominant role ofSCARECROW-like proteins (SCLs) in root biology is associated with auxin,quantitative RT-PCR was used to examine the expression of BnSCL1 gene infour-leaf stage- and 10 dpg-seedlings treated with the synthetic auxin2,4-D. As shown in FIG. 27, BnSCL1 mRNA accumulation increased byapproximately 50% within 30 min of application of 1 mM 2,4-D), and thendecreased rapidly to a lower level, when compared to untreated plants(FIG. 27).

Auxin levels are known to modulate the degradation rate of Aux/IAA(auxin/indole-3-acetic acid protein) family members through aproteolytic regulation mechanism (Zenser et al., 2001). To examinewhether auxin levels also influences the expression pattern of BnSCL1gene, quantitative RT-PCR was used to analyse total RNA isolated fromshoots and roots of 10 dpg seedlings treated with variableconcentrations of 2,4-D ranging from 1 pM to 1 mM (FIG. 28). Expressionof BnSCL1 in shoots was rapidly downregulated by auxin even at thelowest level (1 pM) of 2,4-D, indicating that BnSCL1 response to auxinis very sensitive (FIG. 28 a). BnSCL1 expression in roots, however, wasupregulated by auxin although application of a higher concentration (100μM) of auxin was required to produce an effect (FIG. 28 b). To determinewhether response of BnSCL1 gene to auxin was due to the exogenousapplication rather than the intercellular auxin synthesis, seedlingswere treated for 24 h with 50 μM of naphthylphthalamic acid (NPA), apolar auxin transport inhibitor, and the expression of BnSCL1 inresponse to auxin was analysed using quantitative One-Step RT-PCR. Ascan be seen in FIG. 28 c, the BnSCL1 mRNA accumulation profiles were notchanged both in shoots and in roots after NPA treatment followed by theapplication of auxin at different concentrations. These results suggestthat the response of BnSCL1 to the application of exogenous auxin wastissue-specific, or the expression of BnSCL1 may be regulated by auxindistribution in plants.

Expression of SCR in apical meristems was found to be controlled bychromatin assembly factor-1 (CAF-1) (Kaya et al., 2001), and auxin geneexpression mutations to be located within an Arabidopsis RPD3-likehistone deacetylase gene, HDA6, using map-based cloning approach(Murfett et al., 2001). However, no alterations in gene expression ofendogenous auxin response genes were detected in the mutants and noeffect of auxin-inducible GUS expression was found after seedlings weretreated with HADC inhibitor sodium butyrate at concentration up to I mMfor 24 h (Murfett et al., 2001). To determine whether BnSCL1 response toauxin is modulated by HDA19, 9 dpg seedlings were treated with 2,4-D atconcentrations ranging from 10⁻⁶ to 10³ μM or treated with 50 mM ofsodium phosphate buffer as control after sodium butyrate treatment for24 h at a concentration of 10 mM. Relative expression was investigatedusing quantitative One-Step RT-PCR to analyze RNA extracted from shootsand roots of seedlings. As shown in FIG. 28, although the expressionpattern of BnSCL1 in response to auxin in shoots was different from thatin roots, the inhibition of histone deacetylase led to the expressionprofiles of BnSCL1 in shoots were similar to those in roots, i.e. theexpression was upregulated by auxin at concentration of 1 pM anddownregulated by auxin at higher concentrations. The fact that HDACinhibition led to the alteration of BnSCL1 expression in response toauxin suggests that the response of BnSCL1 to auxin is modulated byhistone deacetylase.

These results suggest a molecular mechanism by which BnSCL1 functions asa transcription factor to regulate gene expression by recruiting HDAC tothe promoter regions of target genes.

Example 6 Modulation of Activity of a Gene of Interest Using aRecruitment Factor

Two constructs are prepared: 1) an activator+reporter construct (FIG.29B) carrying the lacZ reporter gene downstream from a Tet operatorsequence (Tet-7X), and the BnSCL1 and VP16 genes encoding a VP16-SCLfusion protein that is able to bind the Tet operator sequence; and 2) aneffector construct carrying the HDA19 gene (FIG. 29B).

The activator+report construct is introduced and expressed in yeastcells, for example MaV203 cells as described in Example 4, to produce areporter yeast. Activity of lacZ product is quantified by measuring theβ-galactosidase activity using chlorophenol red-β-D-galactopyranoside(CPRG) (GibcoL BRL). In the reporter yeast, expression of theactivator+reporter construct results in the expression of the VP16-SCLfusion protein that binds to the Tet operator sequence, therebyactivating expression of the LacZ reporter gene due to VP16.

The reporter yeast expressing the activator+reporter construct istreated with tetracycline. Expression of lacZ reporter gene isquantified by measuring the β-galactosidase activity using chlorophenolred-β-D-galactopyranoside (CPRG). The expression of theactivator+reporter construct in the presence of tetracycline in yeastcells produces a baseline level of LacZ activity.

The effector construct is then introduced into the reporter yeast sothat the activator+reporter and the effector constructs are bothexpressed, and the activity of the LacZ product determined as indicatedabove. Results demonstrate that LacZ activity is reduced in the yeastexpressing both the activator+reporter and the effector constructs, whencompared to LacZ activity determined in the reporter yeast expressingonly the activator+reporter construct, and approximates the level ofactivity of LacZ activity produced by the reporter yeast when treatedwith tetracycline.

This result indicate that the expression of a gene of interest (in thiscase LacZ) may be reduced by targeting a recruitment factor, for exampleSCL1, to the nucleotide sequence encoding the gene of interest, andpermitting the recruitment factor to bind an HDAC.

A similar set of assays is carried out comprising three constructs: 1) areporter construct carrying the lacZ reporter gene, 2) an activatorconstruct carrying the BnSCL1 and VP16 genes, and 3) an effectorconstruct carrying the HDA19 gene (see FIG. 29A). The constructs areexpressed in yeast cells, for example MaV203 cells as described above inExample 4, in the following combinations:

reporter construct alone,

reporter and activator constructs,

reporter, activator and effector constructs.

The expression of lacZ reporter gene is quantified by measuring theβ-galactosidase activity using chlorophenol red-β-D-galactopyranoside(CPRG) (GibcoL BRL).

The expression of the reporter construct alone in yeast cells produces abaseline level of β-galactosidase activity. Expression of both thereporter and activator constructs yields an elevated level ofβ-galactosidase activity, when compared with the activity observed inthe presence of the reporter construct alone, while the reporter,activator and effector constructs together results in approximatelybackground levels of β-galactosidase activity.

All citations are herein incorporated by reference.

The present invention has been described with regard to preferredembodiments. However, it will be obvious to persons skilled in the artthat a number of variations and modifications can be made withoutdeparting from the scope of the invention as described herein.

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1. An isolated nucleic acid sequence encoding the sequence of BnSCL1 asset forth in SEQ ID NO:81.
 2. The isolated nucleic acid sequenceencoding amino acids 1 to 217 of SEQ ID NO:81.
 3. The isolated nucleicacid sequence of claim 2, encoding amino acids 1 to 358 of SEQ ID NO:81.4. The isolated nucleic acid sequence of claim 2, encoding amino acids 1to 261 of SEQ ID NO:81.
 5. The isolated nucleic acid sequence encodingamino acids 146 to 358 of SEQ ID NO:81.
 6. A construct comprising theinsolated nucleic acid of claim 1, operatively linked with a regulatoryregion.
 7. A construct comprising an isolated nucleic acid operativelylinked with a regulatory region, wherein the isolated nucleic acid isselected from the group consisting of the nucleic acid sequence encoding1 to 358 of SEQ ID NO:81, the nucleic acid sequence encoding 1 to 261 ofSEQ ID NO:81, the nucleic acid sequence encoding 1 to 217 of SEQ IDNO:81, and the nucleic acid sequence encoding 146 to 358 of SEQ IDNO:81.
 8. The construct of claim 6, wherein the isolated nucleic acidfurther comprises a nucleic acid sequence encoding DNA binding protein.9. The construct of claim 7, wherein the isolated nucleic acid furthercomprises a nucleic acid sequence encoding DNA binding protein.
 10. Atransgenic plant comprising the construct of claim
 6. 11. A transgenicplant comprising the construct of claim
 7. 12. A transgenic plantcomprising the construct of claim
 8. 13. A transgenic plant comprisingthe construct of claim 9.